2006 Opening Session Program

September 25

08:00 - 09:15 Registration

09:15 - 09:25 Welcoming notes

09:25 - 10:25 Keynote presentation
Modular regulation of RNA in the brain
Robert B. Darnell, MD, PhD
Howard Hughes Medical Institute, The Rockefeller University

We review here the development and results emanating from a systematic dissection of the function of RNA binding proteins. The paradigm used to approach this problem has been the study of Nova, a neuron-specific RNA bidning protein targeted in an autoimmune neurologic disorder associated with cancer. We have developed a combination of biochemical, genetic and bioinformatics methods to generate a glo bal understanding of Nova’s role as a splicing regulator. Genome-wide idnetificaiton and validation of Nova target RNAs has yielded unexpected ininsights into the protien’s mechaims of action, its role in neurobiology, and, most generally, into the unique roles RNA binding proteins play in the biology of the neuronal synapse. These studies provide us with a paradigm for understanding the role of RNA binding proteins in neurons and in disease, and more genrally, the hoep that it is feasible to develop comprehensive understanding of the regulation of RNA metabolism by regulatory factors.

10:25 - 10:45 Coffee break

10:45 - 10:55 Introduction to catalytically active RNAs

Session 1:


Host: Jean-Pierre Perreault

10:55 - 11:25
Specific on/off adaptor for various nucleic acid enzymes.
F.P. Brière, D. Lévesque and J.P. Perreault
Département de biochimie, Université de Sherbrooke

Many efforts have been made toward the development of gene-inactivating systems using nucleic acid-based drugs. Ribozymes and siRNA are very promising in this regard. Unfortunately, the limited substrate specificity of these approaches is considered today to be a significant hurdle in their development as molecular tools. We have engineer a ribozyme possessing a new biosensor module that switches the cleavage activity from “off” – a safety lock – to “on”, solely in the presence of the appropriate RNA target substrate. Both proof-of-concept and the mechanism of action of this man-made riboswitch have been demonstrated using ? ribozymes. To our knowledge, this Specific On/ofF Adapter (SOFA) is the first report of a ribozyme bearing a target-dependent module that is activated by its RNA substrate, an arrangement which greatly diminishes non-specific effects. Moreover, we have been able to both develop different configuration of the SOFA and adapt the concept on several other nucleic acid-based systems, including cleaving hairpin and hammerhead ribozymes as well as the 10-23 deoxyribozyme. Moreover, the SOFA concept was also adapted to both a hairpin ribozyme ligating two short RNA substrates and a ribozyme catalyzying the 5’-end capping of a RNA strand. The SOFA module is versatile and can be adapted to many projects that have be prematurely aborted. This new approach provides a highly specific and improved tool with significant potential for application in the fields of both functional genomics and gene therapy.

11:25 - 11:55
NMR studies of the Neurospora VS ribozyme
Patricia Bouchard, Geneviève Desjardins, Dean Campbell, Philipe Lampron et Pascale Legault
Université de Montréal,University of Georgia

We are studying the Neurospora VS ribozyme by NMR spectroscopy to gain a better understanding of the structural basis of RNA function. The VS ribozyme is an ideal model system to study structure-function relationships in RNA. Its secondary structure contains six helical domains: stem-loop I forms the substrate domain and stem-loops II-VI constitute the catalytic domain. Although tertiary structural models have been proposed, there is no reported high-resolution structure of the full ribozyme. Substrate recognition by the VS ribozyme depends on an essential loop-loop interaction between stem-loop I of the substrate and stem-loop V from the catalytic domain. We have solved the NMR structures of stem-loop I and stem-loop V fragments. We are currently determining a high-resolution structure of the stem-loop I / stem-loop V complex by NMR spectroscopy to understand the unusual mode of substrate recognition by the VS ribozyme. It has been proposed that the active site of the ribozyme is located in stem-loop VI, and we are studying the structure of a stem-loop VI fragment to better understand the catalytic mechanism of this enzyme. The structure of small RNA fragments such as stem-loops I, V and VI can be determined straightforwardly by standard NMR methods. NMR studies of larger RNAs (> 50 nt) is more challenging. However, novel NMR approaches such as residual dipolar coupling restraints have proven successful for determining global fold of such RNAs. We are using similar approaches to help define the global fold of the catalytic domain of the VS ribozyme.

11:55 - 12:30 Alumni of the year award
Trapped water molecules are essential to structural dynamics and function of a ribozyme
Maria M. Rhodes1, Kamila Reblova2, Jiří Šponer2 and Nils G. Walter1
1- Department of Chemistry, Single Molecule Analysis Group, University of Michigan, 930 N. University Avenue, Ann Arbor, MI 48109-1055;
2- Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic

Ribozymes are catalytically competent examples of highly structured non-coding (nc)RNAs, which are ubiquitous in the processing and regulation of genetic information. Combining explicit-solvent molecular dynamics simulation and single molecule fluorescence spectroscopy approaches, we find that a ribozyme from a sub-viral plant pathogen exhibits a coupled hydrogen bonding network that communicates dynamic structural rearrangements throughout the catalytic core in response to site-specific chemical modification. Trapped long-residency water molecules are critical for this network and only occasionally exchange with bulk solvent as they pass through a breathing interdomain base stack. These highly structured water molecules line up in a string that may potentially also be involved in specific base catalysis. Our observations suggest important, still underappreciated roles for specifically bound water molecules in the structural dynamics and function of ncRNAs.

12:30 - 14:30 Lunch

13:30 - 14:30 Company presentations

14:30 - 14:40 Introduction to RNase III and miRNA synthesis

Session 2:

RNase III-dependant RNA processing and degradation

Host: Sherif Abou Elela

14:40 - 15:10
Biochemical and Genetic Analysis of Yeast RNase III
Ge, D., Larose, S, Ghazal, G., Lamontagne, B., and Sherif Abou Elela
Université de Sherbrooke

Double stranded RNA (dsRNA) is an important signal influencing critical cell functions. Abnormal quantities of dsRNA in the cell inhibit the expression of specific genes (RNAi), suppress translation, and induce interferon response. We are beginning to recognize the dsRNA effects on cell function but we do not know how they are regulated. The goal of our lab is to understand the role of dsRNA-specific nucleases in regulating specific cellular activity such as RNAi, RNA processing, or interferon response. We are using the yeast orthologue of the bacterial dsRNA specific RNase III (Rnt1p) as a model to understand the biochemical and biological activities of eukaryotic RNase III. Experiments that describe Rnt1p substrate specificity and its role in mRNA processing and degradation will be presented.

15:10 - 15:40
Dicer, hepatitis C virus and human immunodeficiency virus
Patrick Provost
Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL – CHUQ, Department of Anatomy and Physiology, Faculty of Medicine, Université Laval.

The ribonuclease (RNase) III Dicer is known to play an essential role in microRNA (miRNA) biosynthesis by converting miRNA precursors (pre-miRNAs) into miRNAs in the vast majority of eukaryotes, including humans. Designed to simulate miRNAs, small interfering RNAs (siRNAs) represent a powerful genetic approach to potently inhibit gene expression by mediating cleavage of the intended mRNA target. This strategy has been applied successfully to suppress replication of several viruses, including hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1). However, evidences that host organisms may defend themselves against viruses came from observations made by plant biologists. Indeed, while investigating the natural antiviral defense mechanism known as posttranscriptional gene silencing (PTGS), Hamilton and Baulcombe (1999) detected the presence of antisense viral RNA of ~25-nt in virus-infected plants by Northern blot. These small RNAs were later found to originate from viral double-stranded RNA (dsRNA) processing by DICER-LIKE 1 in Arabidopsis thaliana. Two human viruses of global importance for human health, HCV and HIV-1, are predicted to encode stem-loop RNA structures resembling miRNA precursors (pre-miRNAs) at their 5’NTR. Therefore, we investigated whether Dicer could recognize and process RNAs derived from HCV and HIV-1, and therefore play a role in host defense mechanisms against these viruses in human cells. I will discuss about the most recent advances in our laboratory that reveal a multifaceted interaction between HIV-1 and a proven antiviral defense mechanism in lower eukaryotes. This project is supported by the Canadian Institutes of Health Research (CIHR).

16:10 - 16:40
Molecular dissection of the small RNAs pathways in C. elegans.
Martin J. Simard
Centre de Recherche en Cancérologie de l'Université Laval, Québec, Canada

Since their discoveries a decade ago, the tiny RNAs have rapidly become the rising star of the RNA world. The small non-coding RNAs precisely regulate gene expression at different levels; they can silence gene transcription, specifically degraded mRNAs fully complementary to their 22 nucleotides long sequences or abrogate protein synthesis. Using the nematode C. elegans as a model system, our group studies the RNA interference and the microRNA pathway, two cellular mechanisms in which small non-coding RNAs play a central role. In order to uncover how RNAi and microRNA pathways control gene expression, we are using diverse biochemical and genetics approaches to identify new cellular components associated to these pathways and to study biological properties of known players such as Dicer and Argonautes proteins. I will present our latest results on the identification and characterization of cellular players in the microRNA and the RNAi pathway and discuss about their potential role in these molecular mechanisms.

16:40 - 17:10
Small RNAs and cancer
Gerardo Ferbeyre
Université de Montréal

Small RNAs are inreasingly impotant in cancer research. A group of natural endogenous RNA called micro RNAs (mirnas) regulate the expression of multiple genes. The expression of some of these mirnas is altered in cancer patients. Our studies focuses in a family of mirnas, the mir17 cluster, able to regulate the expression of E2F1, one important component of the cell cycle regulatory engine and a well-known inducer of apoptosis. We have discovered that the mir17 cluster is a transcriptional target of E2F, providing a regulatory feed-back loop between E2F and these mirnas. Another group of small RNAs, called small hairpins RNAs (shRNAs) are artificially designed as a tool for genetic research in mammalian cells. We are using libraries of shRNAs to discover genes that regulate cell death. We will present the results of one of our screening experiments in osteosarcoma cells.

17:10 - 18:10 Poster competition IA: (Poster # 1-45)

18:10 - 19:10 Poster competition IB: (Poster # 46-89)

19:10 - 20:00 Open posters presentations (Poster # 1–45)

20:00 - 21:00 Dinner

21:00 - 21:30 After dinner speaker
Life as a Scientific Editor
Dorit Zuk

September 26

08:30 - 08:40 Introduction to eukaryotic gene regulation

Session 3:

Regulation of gene expression

Host: Benoit Chabot

08:40 - 09:10
CARM1, alternative splicing and the Survival of Motor Neuron protein: the missing link?
Jocelyn Cote1, Donghang Cheng2 and Mark T. Bedford2
1- University of Ottawa, Faculty of Medicine, Department of Cellular and Molecular Medicine, Center for Neuromuscular Disease, Ottawa, ON, Canada, K1H 8M5
2- The University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, P.O. Box 389, Smithville, TX 78957, USA.

Arginine methylation is a post-translational modification that is often found in RNA binding proteins harboring glycine- and arginine-rich (GAR) domains. Interestingly, SMN, the causative gene for spinal muscular atrophy (SMA), was recently found to interact preferentially with proteins harboring symmetrical dimethylated arginines. This specific form of arginine methylation is performed by Type II methyltransferases (PRMTs), and indeed, at least one of these enzymes is known to regulate SMN activities. CARM1 (a Type I PRMT), does not methylate GAR domains, but rather prefers isolated arginine surrounded by an ill-defined consensus. CARM1 is recruited by many transcription factors as a positive regulator and its activity is known to be important for neuronal as well as skeletal muscle differentiation. Since the neuromuscular system is specifically affected in SMA patients and considering the link between arginine methylation and SMN, we sought to identify novel substrates for CARM1. Using a small-pool screening approach, we identified splicing factors CA150, SF3b4, SmB and U1C. Strinking, we show that CA150 interacts with the tudor domain of SMN, in a CARM1-dependent fashion. This led to the identification of a novel high-affininty binding surface for SMN and suggest that Type I PRMTs should also be considered as potential regulators of SMN activities. Finally, experiments using splicing reporters as well as endogenous target pre-mRNAs reveal that CARM1 promotes exon skipping in an enzyme-dependent manner. Thus, one way in which CARM1 may function is to regulate alternative splicing, by methylating specific splicing factors, hence modulating there interaction with SMN.

09:10 - 09:40
Gene expression in programmed cell death: the TAF6delta pathway
Brendan Bell
Université de Sherbrooke

TFIID is a multi-protein complex that plays a central role in the regulation of expression of protein-coding genes. TFIID is composed of TATA-binding protein together with 14 TBP-associated factors (TAFs). Previous work from our lab suggested that a specialized splice variant of TAF6, TAF6delta, plays an important role in regulating gene expression patterns in certain forms of programmed cell death (apoptosis). TAF6delta is induced in certain forms of apoptosis and replaces the core TFIID subunit TAF6alpha resulting in altered gene expression. We have developed antisense 2’-O-Methyl phosphorothioate RNA oligonucleotide that when transfected into cell lines enforce splice site selection of endogenous TAF6delta. Using this antisense approach we show that TAF6delta expression induces apoptosis. We have further shown that TAF6delta-induced cell death does not require p53, a tumor suppressor protein that interacts directly with TAF6. To determine the outcome of TAF6delta expression at the transcriptome level we have combined antisense RNA with genome-wide microarray studies. The microarray results reveal that TAF6delta expression drives changes in gene expression including the induction of several pro-apoptotic genes, tumor suppressor genes, and genes implicated in angiogenesis. Together our results suggest that TAF6delta alternative splicing is a novel regulatory event that can control cell life or death decisions by altering gene expression programs in response to cell signals.

09:40 - 10:10
RNA Recognition by the Vts1 SAM Domain
Philip Johnson
Department of Chemistry York University

The putative post-transcriptional regulator Vts1p from Saccharomyces cerevisiae, and its related protein Smaug, from Drosophila melanogaster, each use a sterile alpha motif (SAM) domain to bind an RNA pentaloop hairpin termed the Smaug Recognition Element (SRE). The use of a SAM domain for RNA binding is unusual as it is a motif typically associated with protein-protein recognition. Here I present the NMR-derived structure of the SRE Vts1p SAM domain bound to a 19 nt RNA hairpin bearing a 5’-CUGGC’-3’ wild type SRE pentaloop sequence as well as the structure of the unbound SRE RNA hairpin. Structural highlights include direct recognition of a partially exposed guanine base at the apex of the loop by a shallow cleft near the fifth helix of the SAM domain and observation of a base pair between the first and fourth positions of the pentaloop that is stabilized by Vts1p binding.

10:10 - 10:30 Coffee break

10:30 - 10:40 Introduction to retroelements

Session 4:


Host: Eric Masse

10:40 - 11:10
Insertion of group II intron retroelements after transcription terminators
Aaron R. Robart, Wooseok Seo and Steven Zimmerly
University of Calgary

Group II introns are ribozymes and retroelements found in bacteria, archaebacteria, mitochondria and chloroplasts. While most group II introns insert site-specifically into targets through retrohoming, one phylogenetic class in bacteria avoids interrupting ORFs altogether, and instead inserts directly after rho-independent transcription terminators (stem-loop followed by U’s). This property violates principles of homing, because a motif is targeted rather than a specific sequence. In order to address the mechanistic basis for insertion after terminators, we have studied a representative intron of this class, B.h.I1 of Bacillus halodurans. The intron-encoded reverse transcriptase (RT) was expressed and affinity purified, and shown to exhibit RT and maturase (splicing) activities. In vitro, the RT binds to unspliced intron transcripts and induces splicing, thereby forming an RNP complex (intron lariat + RT), that is competent for a subsequent mobility reaction. The RNP is capable in vitro of invading DNA targets having terminator motifs. Analogous to other group II introns, the intron reverse splices into the top strand of the DNA target, and then the inserted intron is reverse transcribed. However, unexpectedly the DNA target must be single-stranded and fold into a structure equivalent to the RNA stem-loop formed during transcription termination. The requirement for a DNA stem-loop in the target explains the specificity for terminator motifs, and also suggests that the process occurs in vivo at single-stranded DNA at either a replication fork or transcription bubble.

11:10 - 11:40
Role of class C group II introns in the formation of mobile gene cassettes in integrons
Paul H. Roy, Grégory Léon, Cecilia Quiroga and Daniela Centrón
Université Laval, Quebec City, Canada and Universidad de Buenos Aires, Argentina

Integrons are elements that use an integron integrase, a tyrosine recombinase, to incorporate gene cassettes into a specific attI site. Mobile cassettes usually consist of a single, promoterless gene accompanied by an attC site, a palindromic sequence of 60-140 bp. The integrase integrates and excises cassettes by attI x attC and attC x attC recombination. In plasmid-borne integrons, where cassettes are mostly antibiotic resistance genes, analysis indicates that the structural genes and their attC sites have evolved separately before being assembled. A major unanswered question is how structural genes become associated with attC sites to form cassettes. Some class C group II introns have been shown to target to transcriptional terminators. Other class C group II introns occur at the junction of a structural gene and an attC site in integrons. These structural gene-group II intron-attC site assemblies are potential intermediates in cassette formation. We have found potential earlier intermediates, structural gene-group II intron and group II intron-attC site, in Nitrosomonas europaea, Geobacter sulfurreducens, and other bacterial genomes. We are investigating how group II introns may capture attC sites (probably by retrohoming to the left end of attC sites, which resemble transcription terminators) and structural genes, then recombine at the DNA level, splice out at the RNA level, and reverse transcribe to yield cassettes. We have demonstrated group II intron mobility and splicing in the S. marcescens and G. sulfurreducens systems.

11:40 - 12:15 Best Seminar Award

12:15 - 12:35 2006 Group Photo

12:35 - 14:10 Lunch

14:10 - 16:00 Group Activity

16:00 - 16:10 Introduction to translation regulation in eukaryotes

Session 5:


Host: Martin Bisaillon

16:10 - 16:40
The ribosomal -1 programmed frameshift in the human immunodeficiency virus type 1.
Léa Brakier-Gingras
Département de biochimie, Université de Montréal

It is known for about 20 years that HIV-1 uses a programmed -1 ribosomal frameshift to synthesise the precursor of its enzymes but the mechanism accounting for this event remains elusive. It has been shown that this frameshift occurs at a homopolymeric slippery sequence followed by a specific two-stem helix, the frameshift stimulatory signal, which controls the frameshift efficiency. However, the precise step of protein synthesis during which the frameshift occurs is controversial. Our assays with a bacterial system support a model in which an abnormal translocation triggers this event. Moreover, assays that measure the efficiency of the HIV-1 frameshift with bacteria as well as with a eukaryotic system demonstrate that the slippery sequence is not restricted to a heptanucleotide, as previously assumed, but is extended on the 5’ site. We also showed that, although the frameshift occurs during the elongation of translation, its efficiency can be altered when the rate of initiation of translation is changed. Finally, this frameshift represents an interesting target to prevent virus replication. The screen of a library of short arginine-rich peptides has enabled us to select candidates that decrease the frameshift efficiency of HIV-1. Such compounds could lead to the development of novel efficient antiviral agents.

16:40 - 17:10
Regulation of the nuclear poly(A) binding protein by arginine methylation in fission yeast
Audrey Perreault, Caroline Lemieux and Francois Bachand
Department of Biochemistry, Université de Sherbrooke, Sherbrooke, Quebec, Canada.

Nearly all eukaryotic mRNAs possess poly(A) tails. Two major poly(A) binding proteins (PABP) bind the poly(A) tract of an mRNA in mammals: PABPC in the cytoplasm and PABP2/PABPN1 in the nucleus. Whereas yeast orthologs of the cytoplasmic PABP are characterized, a gene product homologous to mammalian PABP2 has yet been identified in yeast. During the course of studies aimed at isolating proteins that copurify with the fission yeast protein arginine methyltransferase 1 (RMT1), an uncharacterized protein (Pab2) with significant amino acid identity to human PABP2 was identified. The product of the S. pombe pab2 gene was not essential for mitotic growth and localized to the nucleus, with enrichment in nuclear dots. Consistent with a functional role in poly(A) tail metabolism, recombinant Pab2 demonstrated specific poly(A) binding in vitro, and importantly, mRNAs from pab2-null cells displayed hyperadenylated 3’ ends. We also showed that arginine residues within the carboxy-terminal glycine/arginine-rich domain of Pab2 are modified by methylation, and that this modification is RMT1-dependent. Whereas the arginine methylated and unmethylated forms of Pab2 behaved similarly in terms of subcellular localization, poly(A) binding, and poly(A) tail length control in vivo; Pab2 oligomerization levels were markedly increase when Pab2 was not methylated. Furthermore, we found that overexpression of Pab2 in fission yeast reduced growth rate and that this growth inhibitory effect was exacerbated in rmt1-null cells. Our results establish Pab2 in the control of poly(A) tail length and support a biological role for Pab2 arginine methylation in the regulation of self-association.

17:10 - 18:10 Poster competition IIA: (Poster # 1-45)

18:10 - 19:10 Poster competition IIB: (Poster # 46-89)

19:10 - 20:10 Open posters presentations (Poster # 46-89)

20:10 - 21:10 Banquet

21:10 - 21:30 The RNA group activities in 2006

21:30 - 22:00 Poster prizes

22:00 - 23:59 Dance

September 27

08:00 - 08:10 Introduction to RNA degradation

Session 6:

New insights into mRNA degradation (Flavor of the year)

Host: Mark Schmitt

08:10 - 08:40
Exploring the Logic of RNA Processing Underlying the Gene Expression Program
Dan Herschlag
Stanford University School of Medicine

Central to biological function and regulation is the gene expression program. Substantial progress has been made in globally identifying transcription factors and their targets. Nevertheless, much less is known at the level of RNA processing, despite the myriad of steps subsequent to transcription and the well-documented interrelationships of these steps. We have begun probing RNA processing at a global level in the yeast Saccharomyces cerevisiae. Critical to the understanding of RNA processing are the proteins that bind RNAs, and yeast contain at least 600 such proteins, more than the number of transcription factors. We have assayed the RNAs associated with ~40 yeast RNA binding proteins. The results indicate that individual RNA binding proteins interact with many RNAs, ranging from dozens to thousands, consistent with the notion of Keene and co-workers that RNA binding proteins serve to coordinate related mRNAs. Our results strongly suggest that there is combinatorial control of RNA function by RNA binding proteins. Further, the logic underlying the coordination by RNA binding proteins, at the broadest level, appears to be at the level of organizing RNAs cytotopically -i.e., by the cellular location that they or their encoded proteins act. These and other approaches to dissect and globally describe RNA processing will be required to understand the emergent properties of cellular systems and, ultimately, to predict, control and manipulate gene expression from a knowledge-based perspective.

08:40 - 09:10
Sequence-based predictive modeling of post-transcriptional regulatory networks
Barrett C. Foat, S. Sean Houshmandi, Wendy M. Olivas, and Harmen J. Bussemaker
Department of Biological Sciences, Columbia University, New York, NY 10027;
Department of Biology, University of Missouri, St. Louis, MO 63121;
Center for Computational Biology and Bioinformatics, Columbia University, New York, NY 10032

The dynamic balance between transcription and mRNA degradation determines the steady-state mRNA abundance for each gene in a genome. We took advantage of the fact that information about the condition-specific activity and sequence-specific affinity of RNA binding regulatory factors is implicitly represented in the steady-state mRNA abundances measured using DNA microarrays. By fitting a model based on a physical description of molecular interactions, we were able to gain quantitative insight into the mechanisms that underlie genome-wide regulatory networks. We developed a novel algorithm, MatrixREDUCE, that predicts the sequence-specific binding affinity of several known and unknown RNA-binding factors and their condition-specific activity, using only genomic sequence data and steady-state mRNA expression data as input. We identified and computationally characterized the binding sites for six mRNA stability regulators in the yeast S. cerevisiae, which include two known RNA-binding proteins, Puf3p and Puf4p. We provide computational and experimental evidence that regulation of mRNA stability by the discovered factors is dynamic and responds to a variety of environmental stimuli. Our computational results suggest that Puf3p functions to destabilize mitochondrion-related transcripts when metabolite repressing sugars are present and in response to the drug rapamycin. We experimentally confirmed these predictions by growing a transformed strain expressing a hybrid mRNA designed to contain a functional Puf3p binding site in different culture conditions and measuring its half-life after a transcriptional shut-off. Our work suggests that regulation of mRNA stability is a pervasive regulatory mechanism that rapidly adapts cellular processes to a changing environment.

09:10 - 09:40
Regulation of mRNA Stability by the Yeast Puf Proteins
Wendy M. Olivas
University of Missouri - St. Louis

The regulation of mRNA stability is often mediated by interactions with elements in 3’ untranslated regions (UTRs), thereby influencing poly(A) tail removal and decapping. My lab has focused on the role of the Puf family of RNA-binding proteins in regulating mRNA stability in yeast. This widely conserved family has multiple members across the eukaryotic kingdom, controlling critical developmental decisions by binding and modulating the metabolism of target mRNAs. The yeast Saccharomyces cerevisiae contains six Puf proteins, providing a good model system to study Puf protein specificity and regulatory function. We first identified COX17 as an mRNA target of Puf3p regulation, and characterized two 3’ UTR elements that are responsible for binding Puf3p and promoting mRNA decay. All Puf proteins contain eight copies of an imperfect repeat sequence, and this repeat domain is sufficient for both RNA binding and decay regulation. Mutational analyses within the Puf3p repeat domain identified specific positions involved in binding specificity, as well as a surface loop required for mRNA decay stimulation. In determining how Puf proteins influence the mRNA decay machinery, the Puf3p repeat domain was found to interact with both deadenylation and decapping factors. Moreover, Puf-mediated regulation of mRNA decay is affected by environmental conditions, such that changes in conditions can rapidly modulate Puf3p activity post-translationally. Finally, we identified mRNA targets of other yeast Puf proteins, including two targets that are regulated in a combinatorial fashion by Puf1p and Puf5p. These results provide key insights into the mechanism of Puf protein control of mRNA stability.

09:40 - 10:10
Nonstop mRNA decay: What happens when the ribosome is at its wits’ end.
Ambro van Hoof
University of Texas Health Science Center at Houston

Gene expression is a complex process that all life forms need to carry out in a precisely controlled fashion. The yeast Saccharomyces cerevisiae and probably most other eukaryotes have two general pathways to degrade mRNA. One of the two pathways of mRNA degradation degrades mRNAs by removal of the cap structure, followed by degradation from the 5’ end. In the second pathway, mRNA decay is carried out by the exosome, a complex containing multiple 3' to 5' exonucleases. One important aspect of mRNA decay is to maintain the overall fidelity of gene expression by preferentially degrading aberrant mRNAs that are made by mistakes during mRNA processing reactions. Examples of aberrant mRNAs that are rapidly degraded are mRNAs that include a premature stop codon, and “nonstop” mRNAs lack a stop codon. Nonstop mRNAs are produced frequently by premature addition of a poly(A) tail, and possibly by a variety of other mechanisms. We have previously shown that nonstop mRNAs are preferentially degraded by the exosome and our ongoing characterization of this pathway will be presented.

10:30 - 11:00
Mechanisms and Regulation of Cytoplasmic Deadenylation, the First Necessary Step on the Pathway of mRNA Decay in Mammalian Cells.
Ann-Bin Shyu
The University of Texas Medical School at Houston,

Deadenylation of the tail is the step that triggers cytoplasmic mRNA degradation in mammals. Interactions among three key players, including cis-elements complexed with their cognate binding proteins, poly(A) nuclease complexes and PABP, determine the rate of deadenylation. However, the regulation of deadenylation and the participating poly(A) nuclease complexes in mammalian cells remains poorly understood. Recently, we showed that PAN2 and CCR4 are major poly(A) nucleases active in the biphasic cytoplasmic deadenylation in mammalian cells. PAN2 initiates deadenylation, and is followed by CCR4-mediated poly(A) shortening, leading to decay of the RNA body. Ectopic expression of PAN3, a functionally indispensable auxiliary factor of PAN2 and also a cytoplasmic poly(A)-binding protein (PABP)-interacting protein, modulates the deadenylating activity of PAN2 in a substrate dependent manner. CAF1, a close partner of CCR4, has a very robust poly(A) nuclease activity in vivo, and CCR4 and CAF1 complement each other's function in cytoplasmic deadenylation. Interestingly, TOB, an anti-proliferative transcription factor that simultaneously interacts with CAF1 and PABP, enhances CCR4/CAF1-mediated poly(A) shortening. Immunofluorescent microscopy studies identify new cytoplasmic foci, containing PAN2/3 and CCR4-CAF1, that co-localize with the RNA-processing-bodies (P-bodies). Blocking cytoplasmic deadenylation with a dominant-negative mutant of CAF1 stabilizes mRNA and reduces the number of P-bodies, suggesting that the deadenylation machinery supplies RNA intermediates necessary for P-body formation. A model has been formulated to describe the control of cytoplasmic mRNA turnover at the deadenylation step, and the relationship between deadenylation and other steps in mammalian mRNA decay.

11:00 - 11:30
Estrogen and Vigilin Control of mRNA Metabolism
Shapiro, D.J., Schoenberg, D.R.1, Goolsby-Flavin K., Mao, C., Dodson, R., Ahn, B and Gipson, S.
Dept. Biochemistry, University of Illinois at Urbana-Champaign
1- Dept. of Molecular and Cellular Biochemistry, Ohio State University

In Xenopus laevis, estrogen stabilizes the mRNA coding for the egg yolk precursor protein vitellogenin and destabilizes albumin mRNA. We identified vigilin/Scp160p/DDP1 as an estrogen-inducible protein that binds specifically to a segment of the vitellogenin mRNA 3'-UTR. Vigilin is a ubiquitous highly conserved protein containing 15 related K-homology (KH) nucleic acid acid binding domains. Binding of vigilin to the vitellogenin mRNA 3'-UTR blocks access of polysomal mRNase I (PMR-1) to 2 cleavage sites in the 3'-UTR and prevents cleavage of vitellogenin mRNA. Vigilin binds specifially to hyperedited inosine-rich RNAs that are retained in the nucleus and plays a role in chromatin silencing. Our studies indicate that intersemgent RNA transfer plays an important role in vigilin's ability to rapidly identify and locate its diverse intracellular RNA binding sites. RNAi-mediated knockdown of human vigilin induces apoptosis and cell death. Microarray analysis indicates that knockdown of human vigilin results in rapid destruction of metal transcription factor MTF-1 mRNA and down-regulation of numerous mRNAs involved in cell growth. Starvation of cells mimics the effect of vigilin knockdown. The potential role of vigilin as an RNA binding protein that controls mRNA degradation and serves as a cell growth sensor will be discussed. Supported by NIH DK50080.

11:30 - 12:30 Student choice lecture
Retrotransposons - gene mobilization through an RNA intermediate
Jef Boeke
Johns Hopkins University School of Medicine

Retrotransposons are found in all eukaryotes and are abundant components of mammalian genomes. The retrotransposon RNA is both a template for protein synthesis (mRNA) and a source of genetic material (genomic RNA). While retroelements are known from prokaryotes, the explosion of retrotransposon types and abunance is clearly correlated with the emergence of eukaryotes, and is probably directly related to distinctly eukaryotic aspects of RNA metabolism such as caps, polyA tails, and other unique aspects of RNA processing machinery such as P-bodies that may have arisen in response to the evolution of nucleocytoplasmic compartmentalization and/or multicellularization. Two distinct classes of retrotransposon are typified by the yeast Ty1 and mammalian L1 elements, although both types are widely distributed phylogenetically in eukaryotes. These classes are evident both structurally and functionally. The former are "retroviral-like" in many ways, and use a classical transposase/integrase based cutting and joining reaction conserved with bacterial DNA transposons, The latter use a reaction called "TPRT" or target primed reverse transcription to insert the element DNA at the new target site. Both classes of elements have had and continue to have a variety of profound effects on the structure of the genomes of their hosts, and on the evolution of these genomes. Experiments on the effects of massively increasing transposon copy number on the host genome will be discussed.

12:30 - 13:30 Light lunch

Poster Sessions

Poster Session 1

1 . Xian Jin Lian*, Olivier Drouin*, Jason Behrman and Imed-eddine Gallouzi
Caspase-dependent HuR cleavage is a key regulatory step of the aging process
McGill University
HuR is an RNA-binding protein that affects the expression of its mRNA targets by affecting their turnover, cellular movement or translation. HuR associates with these messages through an AU-rich sequences (AREs) found in their 3' untranslated region (3'UTR). Previous studies have shown a link between senescence and HuR expression as well as its effect on many ARE-containing mRNAs that encode for key cell cycle players. It has been shown that senescence involves a cell-cycle arrest mediated by the overexpression of the cyclin kinase inhibitor p21cip1. Senescence also represents one of the first regulatory steps leading to cellular aging. Since the cellular localization of HuR protein and its association with protein partners have been shown in other systems to modulate its activity, we investigated if they affect HuR's implication in the aging process. Using western blots and immunofluorescence approaches on human fibroblasts induced for senescence (IDH-4), we observe that HuR, which is mainly nuclear in young cells, translocates partially to the cytoplasm during the aging process. Furthermore, we also showed that HuR protein is cleaved in a caspase-dependent manner before senescence is induced. HuR cleavage products seem to play a crucial role in the aging process. Indeed, using both non-cleavable and cleavage products of HuR, we show that HuR cleavage is essential for conservation of the "young" phenotype. Conversely, the non-cleavable (D226A) isoform of HuR, which localizes only in the nucleus, leads to an increase in the p21cip1 expression as well as the activation of the aging process. RNAi-mediated HuR depletion results in a significant decrease in the steady state levels of p21cip1 mRNA and protein only in young cells. Together our data suggest that HuR regulates the expression of p21cip1 at early stages of the aging process and its caspase-dependent cleavage is likely to be required to maintain the young phenotype.
2 . Sahar Soltanieh, Vincent Lemay, Frédéric Guérin and François Dragon
The putative RNA helicase Dbp4 is required for SSU processome function
Département des sciences biologiques Centre de recherche BioMed Université du Québec à Montréal Montréal (Québec), Canada
Dbp4 is a putative RNA helicase of the DEAD-box family. This enzyme is phylogenetically conserved and essential for growth of the yeast Saccharomyces cerevisiae, indicating its critical function in the cell. Dbp4 was identified in a multicopy suppressor screen designed to isolate proteins that interact with the U14 snoRNA (1). This genetic interaction suggested a role for Dbp4 in ribosome biogenesis because U14 is involved in processing reactions that lead to the production of 18S rRNA. A recent study revealed that Dbp4 is necessary to release U14 from the pre-rRNA (2), and that depletion of Dbp4 impaired early pre-rRNA cleavages at sites A0, A1 and A2. However, the defect at site A0 is not consistent with U14 function, which is only required for cleavages at sites A1 and A2. We performed immunoprecipitation experiments to examine the physical interaction between Dbp4 and U14. The U14 snoRNA did not co-immunoprecipitate with Dbp4, however, the U3 snoRNA and the U3-specific protein Mpp10 were detected in the immunoprecipitates. Because Mpp10 is required for U3 function these results suggest that Dbp4 is associated with functionally active U3. This nucleolar particle, termed the SSU processome, sediments at ~80S in sucrose gradients (3). We show that Dbp4 co-sediments with the U3 snoRNA at ~80S, further indicating that Dbp4 is directly associated with the SSU processome. This association is probably transient because only a small fraction of the U3 snoRNA and Mpp10 were detected in immunoprecipitates. Taken together our results are consistent with a role for Dbp4 in SSU processome function in the three early cleavages at sites A0, A1 and A2. (1) Liang, Clark & Fournier (1997) Mol. Cell. Biol. 17: 4124-4132. (2) Kos & Tollervey (2005) Mol. Cell 20: 53-64. (3) Dragon et al. (2002) Nature 417: 967-970. Supported by the Natural Sciences and Engineering Research Council of Canada
3 . Virgine Dormoy-Raclet*, Isabelle Ménard*, Éveline Clair, Rachid Mazroui and Imed Eddine Gallouzi
The RNA-binding protein HuR interacts with a U-rich element within the beta-actin 3'UTR to regulate its stability
McGill Unversity
The regulation of mRNA turnover is controlled by specific interactions between cis-acting elements and trans-acting factors. An important cis-acting element is the adenylate-uridylate-rich element (ARE) found in the 3' untranslated regions (3' UTRs) of many labile mRNAs. HuR is a ubiquitously expressed RNA binding proteins that specifically interacts with and stabilizes ARE-containing mRNAs. Using RNA interference to knockdown the expression of HuR in HeLa cells, we demonstrate that HuR plays an important role in the maintenance of <=-actin mRNA stability. We show that HuR knockdown leads to a decrease in the steady-state levels of <=-actin mRNA and protein, as well as identify an Uridylate-rich (U-rich) element within the 3'UTR of <=-actin mRNA as the HuR binding site. HuR knockdown leads to a decrease of cell growth and to a disorganization of stress fibers in HeLa cells. We demonstrate that HuR deficiency causes impairments in adhesion, migration and invasion and that these defects correlate with defective formation of cytoskeletal structures, namely actin stress fibers.
4 . Olivier Drouin, Eveline Clair and Imed Gallouzi
Dual role of HuR in cell stress response
McGill University
HuR is an RNA-binding protein regulating different post-transcriptional events of target mRNAs containing adenosine (A)- and uridine (U)- rich (AREs) regions in their 3'untranslated region (3'-UTR). ARE-containing mRNAs are labile and their half-lives increases through association with HuR protein. In response to stress, HuR has been shown to mediate apparently antagonistic actions by activating both pro- and anti-apoptotic processes. We and others have demonstrated that under different assaults HuR stabilizes the message encoding for the proapoptotic factor, caspase-9 and regulates the translation of the antiapoptotic protein prothymosin alpha. Therefore, we hypothesize that HuR's implication in the stress response depends not only on cell type but also on the type and duration of the stress applied. Using Northern blot analysis combined to immunopreciptiation and RT-PCR techniques, we demonstrate that soon upon a stress is applied HuR associates with both pro- (procaspase-9) and anti-death messages (survivin). However, if the stress is unsustainable and cell death is engaged, HuR associates only with proapoptotic messages such as procaspase-9 increasing its half-life. Thus, the preferential association of HuR with different subset of stress response-related transcripts seems to be sequential. Therefore, we propose that HuR acts as a sensor of stress in cells, switching from anti- to pro-apoptotic mRNAs depending on the cell's stress inputs.
5 . Audrey Dubé and Jean-Pierre Perreault
Identification of proteins interacting with Peach Latent Mosaic Viroid RNAs
Département de biochimie, Université de Sherbrooke
Viroids are small (300-400 nucleotides), single-stranded and circular RNAs which can infect higher plants and induce diseases. Plants infected with viroids display chlorosis, their growth can be affected and in some cases the infection leads to the death of host plant. These non-encapsidated RNA do not code for any proteins and seem to be dependent on host for their replication or any other function. Both, phenotypic (e.g. self-catalytic activities) and genotypic characteristics of viroids, have led them to be described as relics from the precellular RNA world. In essence, viroids constitute a minimal biological system ideal for detailed RNA structure/function analyses. Peach latent mosaic viroid (PLMVd) is a viroid from the Avsunviroidae family which infect peach trees and other plants of the rosaceae family. Those pathogenous RNAs replicate in the chloroplast of their host cells using a symmetric rolling circle mechanism. Our general goal is to identify host proteins that interact with PLMVd and characterize these interactions. We partially purified host proteins from infected peach leaves by affinity HiTrap Heparin Sepharose HP column. We detected RNA-protein interactions by Northwestern hybridizations and identified eight proteins by mass spectrometry (LC-MSMS). Partial characterization of these interactions using recombinant proteins and the PLMVd RNA will be presented. All this work should lead a better understanding of PLMVd’s life cycle.
6 . Julie Motard and Jean-Pierre Perreault
Development of a RNAp dependent rolling circle replication
Université de Sherbrooke
Viroids, single-stranded circular RNA molecules, infect higher plants without coding for any known protein. However, they must replicate in host cells through an RNA polymerase DNA dependant (RNAp) via a rolling circle mechanism. Escherichia coli RNAp has been shown to replicate the Peach Latent Mosaic Viroid (PLMVd) in vitro, although inefficiently. In order to overcome this hurdle, we would like to isolate a more efficient RNA promoter for the E. coli RNAp. A circular randomized template protocol has been designed for this purpose. The product contains a hammerhead self-cleaving motif giving a monomer. To date, all of the steps have been verified and optimized with a model control based on the minimal replication domain of PLMVd. Currently, we are performing the selection work. This methodology should reveal more efficient promoters than those identified in viroids. Eventually, it may lead to the development of an efficient RNA rolling circle replication that would be practically autonomous, whose only outside requirement would be for an RNAp.
7 . Meredith McLaren and Alan Cochrane
Effects of Sam68 on HIV-1 RNA processing
Department of Medical Genetics and Microbiology, University of Toronto
HIV-1 Rev functions to export incompletely spliced viral transcripts from the nucleus, allowing expression of the structural and enzymatic proteins of the virus. Co-expression of the nuclear factor Sam68 (Src-associated during mitosis) results in potent stimulation of HIV-1 gene expression. Previously, we showed that overexpression of Sam68 increased the proportion of unspliced 3’ end processed viral RNA without enhancing Rev-dependent export of this RNA. We also showed that overexpression of the GSG domain alone could enhance 3’ end formation of unspliced viral RNAs in the presence of Rev. Current experiments are aimed at further defining the region of Sam68 required for enhancement of 3’ end formation of HIV-1 and doublesex derived transcripts. Experiments to determine whether endogenous Sam68 is required for proper 3’ end cleavage of unspliced viral RNA are being performed, the results of which will be presented.
8 . Marie Soulière, Martin Bisaillon and Jean-Pierre Perreault
Kinetics and mechanism of action of a mRNA capping enzyme.
Université de Sherbrooke
Eukaryotic messenger RNAs harbor a protective cap structure at their 5’-end that is essential for stability, splicing, transport and translation. The cap is synthesized by three enzymatic steps, the second being catalyzed by an RNA guanylyltransferase. This activity operates via two main reactions, the first producing a covalent enzyme•GMP intermediate before the GMP moiety is tranferred to a diphosphorylated mRNA 5’-end. The smallest known RNA guanylyltranferase stems from the A103R gene of Paramecium bursaria Chlorella virus – 1. Radioactive and fluorescence kinetics studies on that protein allowed the identification of reaction intermediates and the quantification of association, dissociation and acticity rates and constants. Moreover, circular dichroism and fluorescence experiments showed thermodynamic and conformational variations between various intermediates. These experiments lead towards the elucidation of the mechanism of action of RNA guanylyltransferases, which could eventually conduct to the development of antiviral or antifungic drugs specific for this essential activity.
9 . Frédéric Picard-Jean and Martin Bisaillon
Characterization of West Nile virus RNA triphosphatase NS3 complexed with RNA.
Université de Sherbrooke
The West Nile virus (WNV) primarly infects birds but occasionally humans where the infection can lead to a fatal brain inflammation. There is still no vaccine or effective therapeutical treatment available against this disease. Like Hepatitis C virus, WNV is part of the Flaviviridae family and contains a capped positive ssRNA genome coding for a large polyprotein presursor. The 5’ cap, which ensures the mRNA stability and enhances the genome translation, is synthesized in part by the non-structural protein 3 which has three enzymatic functions including an RNA triphosphatase activity. Since this triphosphatase is significantly different from its human holomog, it represents a very potent pharmalogical target. The present study presents a quantative characterization, using fluorescence spectoscopy, of the interaction between NS3 and the RNA. The results such as minimal RNA length required for interaction, real-time binding kinetics, thermodynamics and effect of the ionic strength on the NS3-RNA interaction leads to a better understanding of the formation of the NS3-RNA complex.
10 . Atef nehdi and Jean-Pierre Perreault
Study and molecular engineering of delta ribozyme by in vitro selection
Département de biochimie, Faculté de médecine, Université de Sherbrooke
In order to revisit the architecture of the catalytic center of the antigenomic hepatitis delta virus (HDV) ribozyme we developed an unbiased in vitro selection procedure that efficiently selected novel variants from a relatively small set of sequences. Using this procedure we examined all possible variants from a pool of HDV ribozymes that had been randomized at 25 positions (4(25)). The isolated set of sequences shows more variability than do the natural variants. Nucleotide variations were found at all randomized positions, even at positions when the general belief was that the specific base was absolutely required for catalytic activity. HDV ribozyme appears to possess a greater number of constraints, in terms of sequences capable of supporting the catalyzed cleavage, than do other catalytic RNAs. This supports the idea that the appearance of this catalytic RNA structure has a low probability, which may explain why to date it has been found in nature only in the HDV. To resolve this low rate of variability in our library of selected ribozyme, we sequenced more than 500 clones. Covariation analysis using only the most efficient variants (ribozymes that cleave at 60% or more) reveals many new interactions within the catalytic core (interaction in the bottom of the P2 stem, in the loop L3, and interaction between the P3 stem and the P1 stem). These interactions should be confirmed by directed mutagenesis, enzymatic and chemical probing as well as by crosslinking.
11 . Grégory Léon and Paul H. Roy
Mobility and activity of the class C group II intron of Geobacter sulfurreducens
Université Laval, Quebec City, Canada
Integrons and gene cassettes are considered the main genetic elements in the evolution of plasmids and transposons that actively participate in the dissemination of genes coding for bacterial resistance mechanisms. In integrons, mobile gene cassettes are excised and integrated by the integron integrase, and usually consist of a single promoterless structural gene associated with an attC site. In plasmid-borne integrons, where cassettes are mostly antibiotic resistance genes, sequence analysis indicates that structural genes and their attC’s have evolved separately before being assembled. A major unanswered question is how structural genes become associated with attC sites to form cassettes. We have found in clinical and environmental strains several examples of group II class C introns associated either with integron cassettes or with isolated attC sites. In both cases, introns were inserted site specifically into the 5’ end of attCs. We have found the latter type, group II intron-attC site without an associated structural gene, in Nitrosomonas europaea, Geobacter sulfurreducens and Shewanella spp.. Bacterial class C introns usually insert after transcriptional terminators rather than into defined homing sites. In some cases the introns are >95% identical, yet have inserted into different attCs, which share only conserved boundaries and a stem-loop structure. We are investigating how group II introns may capture attC sites and structural genes and recombine to assemble them. We have demonstrated the splicing, reverse transcriptase and retrohoming activities of the G. sulfurreducens intron. Certain bacterial group II introns are thus mobile elements with affinity for several resistance cassette attC sites.
12 . Renaud Tremblay, Jean-François Lemay and Daniel A. Lafontaine
Core requirements of the adenine riboswitch aptamer for ligand binding
Département de biologie, Faculté des sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada
The adenine riboswitch aptamer, the A box, positively regulates gene expression upon adenine binding. To provide insight into structure-function relationships, important for the adenine riboswitch aptamer, we have created alignments for 6 aptamer sequences that reveal the core requirements. In addition, 2-aminopurine (2AP) binding studies have been used to test the consensus sequence derived from the alignment. Overall, the consensus secondary structure is consistent with 2AP binding studies. However, a position in the core, previously identified as variable, shows restriction in nucleotide sequence. Furthermore, this restriction is found to be related with the ligand specificity of the riboswitch. The implications of this relationship for the riboswitch gene regulation mechanism are discussed.
13 . Annie D'Amours, Bruno Lamontagne & Raymund J. Wellinger
Characterisation of the Secondary Structure of a Potential Pseudoknot in the TLC1 Telomerase RNA Of S. cerevisiae.
Département de Microbiologie et Infectiologie Faculté de Médecine Université de Sherbrooke
In eukaryotes, the synthesis of chromosome ends, the telomeres, depends on an enzyme called telomerase. This particular ribonucleoprotein is composed of a reverse transcriptase subunit, Est2p, a number of other proteins, as well as an RNA moiety, TLC1 in yeast, which dictates telomere repeat addition. Results derived from many organisms have shown that the association of the telomerase RNA to the holo-enzyme relies much more on the secondary and tertiary structure of the RNA than sequence specificity. In particular, we recently obtained evidence suggesting the presence of a pseudoknot near the template region of the yeast telomerase RNA1. The region of this potential pseudoknot overlaps with the region of Est2p association and given the presence of a pseudoknot structure in or near the catalytic core of telomerase of many organisms, a pseudoknot could be a common structural theme important for some aspect of telomerase function2. However, the presence of the pseudoknot in the telomerase RNA of S. cerevisiae has not yet been firmly established. Therefore, our goal is to establish a secondary structure in this particular region of the RNA using biochemical approaches. We constructed a shortened version of the catalytic core RNA and use RNase mapping for this purpose. Here we will present the first mapping results derived from this approach and describe our future plans for this project. Refs: 1. Dandjinou, A.T. et al. 2004. Curr Biol. 14: 1148-58. 2. Chen, JL. and Greider, CW. 2004. Proc Natl Acad Sci USA. 101: 14683-4.
14 . Vincent Lemay and François Dragon
Identification of novel proteins associated with snR30, a processing snoRNP of the H/ACA class.
Département des sciences biologiques, Centre de recherche BioMed, Université du Québec à Montréal
Most H/ACA snoRNPs are involved in pseudouridylation of rRNA, however no target has been found for snR30. On the other hand, snR30 is required for the early processing reactions that lead to the production of 18S rRNA in the yeast Saccharomyces cerevisiae. We hypothesized that, in addition to the four common H/ACA proteins Cbf5 (dyskerin), Gar1, Nhp2 and Nop10, snR30 might contain specific proteins that contribute to its unique functional properties. To test this hypothesis we devised a two-step affinity purification strategy to selectively purify snR30 snoRNP and identify its proteins components. The S1 aptamer, an “RNA tag” that has high affinity and specificity for streptavidin, has been introduced in the snR30 snoRNA and used as a “hook” to isolate the tagged RNP with streptavidin-linked agarose beads. This construct was expressed in a strain expressing TAP-tagged Gar1, a common H/ACA protein. Our results indicate that aptamer-tagged snR30 snoRNA is functional since plasmid-borne S1-snR30 can complement a strain depleted of endogenous snR30. Northern blotting and pCp labeling experiments showed that S1-tagged snR30 snoRNA can be selectively purified following our two-step procedure. Proteins associated with S1-snR30 were fractionated on SDS-polyacrylamide gels, and subjected to immunoblotting and silver staining. These experiments respectively revealed that aptamer-tagged snR30 RNA is associated with H/ACA proteins Cbf5p and Nhp2p, and a number of non-identified proteins. Mass spectrometry analyses confirmed the presence of Cbf5, Gar1, Nhp2 and Nop10. We are currently proceeding with analyses on the other proteins to determine if they are true constituents of snR30.
15 . Benoit Heppell and Daniel A Lafontaine
Thermodynamic analysis of a SAM riboswitch aptamer
Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke.
Riboswitches are novel discovered RNA molecules that are part of mRNA and that consist of a sensor domain able to bind a cognate ligand with high affinity and specificity. To control gene expression, SAM riboswitch must exist under two forms : ON state or OFF state, which is determin by the stability of the complex. In this work we study the global stability of the aptamer under various conditions and we extract thermodynamical parameters, using Van’t Hoff law, from UV spectroscopy thermal curves. Firstly, because the stem P1 determines the switch between ON and OFF states, we study the influence of P1 lenght on global stability. Enthalpy changes (?H) indicate that the stability of the P1 stem influences the stability of aptamer. Formation of the complex SAM-RNA increases the ?H suggesting that SAM binding stabilizes the RNA structure. Secondly, native gel and fluorescence studies using 2-aminopurine show the presence of a K-turn motif in the stem P2 and a pseudoknot interaction between the loop P2 and the junction 3/4. Van’t Hoff enthalpy and gel shift assays for both pseudoknot and K-turn mutants indicate that both motifs influence the stability of apatmer. Fluorescence and temperature melting analysis show that binding of L7Ae protein to K-turn help the aptamer to fold and therefore stabilizes the aptamer. In conclusion, the K-turn, pseudoknot, stem lenght and ligand binding are important for the stability of the aptamer and thus, can influence the equilibrium between ON or OFF state.
16 . Geneviève Pépin, Marija Rakonjac, Andreas Hermansson, Isabelle Goulet, Johanne Doucet, Bengt Samuelsson, Olof Rådmark and Patrick Provost
Human 5-lipoxygenase interacts with Dicer double-stranded RNA binding domain
Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL – CHUQ, Department of Anatomy and Physiology, Faculty of Medicine, Université Laval and Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77, Stockholm, Sweden.
5-Lipoxygenase (5LO) is an enzyme that catalyzes the first two steps in the biosynthesis of leukotrienes, which are potent mediators of inflammation. In human, 5LO expression is restricted to polymorphonuclear leukocytes, monocytes/macrophages, mast cells, B-lymphocytes, dendritic cells, and foam cells of atherosclerotic tissues. The lipoxygenase activity of 5LO is believed to reside within its iron-containing C-terminal catalytic domain, whereas its C2-like N-terminal ß-sandwich binds Ca2+, leading to Ca2+ stimulation of enzyme activity. Designed to identify cellular proteins that could interact with 5LO, a yeast two-hybrid screen led to the identification of partial cDNAs encoding human Dicer C-terminus. We explored further this interaction and delineated the shortest 5LO-interacting domain to the Dicer dsRBD. 5LO interacted directly with the Dicer dsRBD in GST binding assays in vitro. Whether 5LO can interact with the Dicer C-terminus in vivo was confirmed by coimmunoprecipitation from transiently-transfected HEK 293 cells. Using a deletional mutagenesis approach, we failed to generate a 5LO mutant that retained Dicer binding properties. However, these were lost upon alanine substitution of Trp residues 13, 75 and 102, arguing for the involvement of the 5LO N-terminal ß-sandwich in Dicer dsRBD binding. Collectively, our results suggest that the Dicer dsRBD may function as a protein interaction module recognized by the non-catalytic, N-terminal C2-like domain of 5LO. Our study also broadens the repertoire of Dicer-interacting proteins and may provide an intriguing link between inflammation and regulation of gene expression. This study is supported by the Canadian Institutes of Health Research (CIHR).
17 . Simon Blouin, Daniel Lafontaine
A kink-turn motif located in the lysine riboswitch aptamer is important for the formation of a loop-loop interaction and for ligand binding
Université de Sherbrooke
Riboswiches are genetic control elements usually found in the 5’-UTR of certain messenger RNAs and they regulate gene expression by ligand-induced structural changes. In Bacillus subtilis, the lysine riboswitch modify its structure by binding of natural ligand L-lysine to stop the lysC gene transcription. The secondary structure of the lysine riboswitch aptamer is organized around a five-way junction that has been shown to undergo structural changes upon ligand binding. The 2-aminopurine (2AP) fluorescent base was introduced in the riboswitch for monitoring structural changes. 2AP fluorescence is increased by the presence of magnesium ions or lysine suggesting that 2AP is exposed to the solvent when the aptamer folds or binds ligand. A loop-loop interaction has been proposed to occur between P2 and P3 loops. A mutational analysis conducted using fluorescent aptamers shows that this interaction is present and important for lysine binding. Upon close inspection of P2 stem, an unconventional kink-turn motif was identified that does not conform to the established k-turn consensus structure. It has been shown that this motif induces a considerable angle in P2 stem and the aptamer was found to form a specific complex by this motif with the L7Ae protein of Archaeoglobus fulgidus in the low nanomolar range. Both effects are inhibited when mutations known to abolish the k-turn structure are introduced. Moreover, fluorescence assays show that the k-turn motif is important for the loop-loop interaction and for lysine binding. The implications of protein binding ability of the riboswitch are being examined.
18 . Isabelle Plante, Dominique L. Ouellet, Corinne Barat, Marie-Ève Janelle, Louis Flamand, Michel J. Tremblay and Patrick Provost
Identification of a novel microRNA derived from HIV-1
Centre de Recherche en Rhumatologie et Immunologie and Research Center in Infectious Diseases, Centre de Recherche du CHUL – CHUQ, Department of Anatomy and Physiology and Department of Medical Biology, Faculty of Medicine, Université Laval.
MicroRNAs (miRNAs) are short 21- to 24-nucleotide (nt) RNA species expressed in most eukaryotes examined to date and generated upon successive processing of stem-loop structured primary miRNAs (pri-miRNAs) and miRNA precursors (pre-miRNAs). We have previously reported the ability of recombinant human Dicer to cleave double-stranded RNAs (dsRNAs) as well as stem-loop structured pre-miRNAs. Searching for medically-relevant substrates of Dicer, we identified the Trans-Activation Response (TAR) element of human immunodeficiency virus type 1 (HIV-1). TAR, which is essential for efficient viral transcription, is a 59-nt stem-bulge-loop RNA structure located at the 5' end of all HIV-1 transcripts found in the nucleus and cytoplasm. We have found that TAR RNA, whose structure resembles that of pre-miRNAs, can be bound and cleaved by recombinant human Dicer into 21- to 24-nt RNA products in vitro. In addition, a small RNA derived from TAR was detected in RNA samples from NL4-3-infected primary human CD4+ T lymphocytes. Using RNase protection assays, we were able to detect a miRNA derived from TAR in cells expressing reporter gene transcripts bearing the HIV-1 long terminal repeat (LTR) at their 5'end. Expression of the HIV-1 TAR-derived miRNA was validated functionally through downregulation of a reporter construct bearing a complementary sequence. Our results support the ability of the ribonuclease III Dicer to recognize and process HIV-1 TAR RNA into miRNAs in vitro and in vivo, thereby suggesting a contributory role of Dicer in host defenses against HIV-1. This study is supported by the Canadian Institutes of Health Research (CIHR).
19 . Francois Bolduc and Jean-Pierre Perreault
Initiation of Replication of the Peach Latent Mosaic Viroïd
Département de Biochimie, Université de Sherbrooke
Viroids infect higher plants and replicate through a rolling circle mechanism in either the nucleus or the chloroplast depending on which family they belong. Since viroïds do not encode any proteins, their replication relies on the host transcriptional machinery and self-catalytic RNA activities. Recent data has demonstrated that DNA-dependent RNA polymerases (DdRPs) could be responsible for the replication of these small RNA molecules. To understand how DdRPs switch from DNA to RNA template, we undertook to identify the initiation site of replication using the Peach latent mosaic viroid (PLMVd) as a model species. This viroïd replicates in the chloroplasts of peach leaves and we took advantage of the fact that nascent transcripts should possess a 5’-triphosphate extremity to identify the initiation sites of both plus and minus polarity-strands. Using PLMVd RNA isolated from infected leaves in combination with RT-PCR, primer extension techniques and the “Terminator enzymatic assay”, we resolved this controversial issue.
20 . Rade Sajic, Kenneth Lee and Alan Cochrane
Inhibition of HIV-1 Replication Through Manipulation of Viral 3' end Processing
Department of Medical Genetics and Microbiology, University of Toronto
The rapid evolutionary rate of HIV-1 has lead to the emergence of multi-drug resistant variants, emphasizing the need for novel inhibitory methods. One such method could be based upon inhibiting viral gene expression through disruption of HIV-1 RNA processing. A means of accomplishing this goal is through use of modified U1snRNA variants that target highly conserved regions of HIV-1 at its terminal exon and prevent 3’ end formation. The modification consists of a 10-nucleotide substitution at the 5’ end complementary to the conserved HIV-1 regions. When modified U1snRNA targeted to HIV-1 were cotransfected with replication deficient HIV-1 proviruses, western and Northern blots indicate a specific and significant reduction in the level of viral RNA expression and protein production. In this study we show that suppression of viral gene expression is dependent on the presence of the U1-70k protein component of the U1snRNP complex, while the loss of the U1-A protein component lead to a minimal loss of the inhibitory effect. A mutant U1snRNA form lacking U1-A binding has potential as a therapeutic agent by maintaining the inhibitory effect while decreasing the potential of cryptic splice site activation in the host genome. To further investigate targeted U1snRNA inhibitory effects on HIV-1, efforts are currently underway to determine if this approach has the ability to suppress protein expression of a broad range of infectious HIV-1 strains. If shown effective, our ultimate goal is to construct a stable CD34+ cell line expressing the U1snRNAs, and test the resultant CD4+ progeny for inhibitory effect.
21 . Jonathan Perreault, Gilles Boire and Jean-Pierre Perreault
Phylogenetic study of the Ro associated Y small RNA genes
Département Biochimie, Faculté de Médecine, Université de Sherbrooke
In this sequencing era, the number of known genomes increases at an incredible pace. In fact, it seems to increase faster than it is analysed. Multiple softwares and computational methods are now necessary to truly take advantage of all the available data. We developped Perl scripts to use local alignment algorythm with 27 animal genomes to study the Y small RNAs. These RNAs are conserved in the animal kingdom and a lot of corresponding pseudogenes are known to occur, especially in primates. We use these retropseudogenes to construct a robust phylogenetic tree of the Y RNA genes. Further evolution insight is provided by the comparison of the Y retropseudogenes across species, which helps understand retrotransposition behaviour of genomes, mostly among mammals and even more among primates. Finally, we studied local sequence variation in the near proximity of the Y pseudogenes, which we complemented with sequencing of a number of pseudogenes in two other primates.
22 . Isabelle Bougie and Martin Bisaillon
Inhibition of a metal-dependent viral RNA triphosphatase by decavanadate
Université de Sherbrooke
Paramecium bursaria Chlorella virus, a large DNA virus that replicates in unicellular Chlorella-like algae, encodes an RNA triphosphatase which is involved in the synthesis of the RNA cap structure found at the 5’ end of the viral mRNAs. The Chlorella virus RNA triphosphatase is the smallest member of the metal-dependent RNA triphosphatases that include enzymes from fungi, DNA viruses, protozoans and microsporidian parasites. In the present study, we investigated the ability of various vanadate oxoanions to inhibit the phosphohydrolase activity of the enzyme. Fluorescence spectroscopy and circular dichroism studies were used to directly monitor the binding of decavanadate to the enzyme. Moreover, competition assays show that decavanadate is a potent noncompetitive inhibitor of the phosphohydrolase activity, and mutagenesis studies indicate that the binding of decavanadate does not involve amino acids located in the active site of the enzyme. In order to provide additional insight into the relationship between the enzyme structure and decavanadate binding, we correlated the effect of decavanadate binding on protein structure using both circular dichroism and guanidium hydrochloride-induced denaturation as structural indicators. Our data indicated that no significant modification of the overall protein architecture is occurring upon decavanadate binding. However, both fluorescence spectroscopy and circular dichroism experiments clearly revealed that the binding of decavanadate to the enzyme significantly decreased the structural stability of the enzyme. Taken together, these studies provide crucial insights into the inhibition of metal-dependent RNA triphosphatases by decavanadate.
23 . Jerome Mulhbacher, Jean-François Lemay and Daniel Lafontaine
Revisiting Guanine Riboswitch
Département de Biologie, Faculté des Sciences, Université de Sherbrooke
Guanine-sensing riboswitches are part of mRNAs and are composed of two structural domains: an aptamer (Gbox) able to bind guanine and an expression platform which performs transcriptional control in function of the guanine concentration. The Gbox is organized around a three-way junction, in which a loop-loop interaction takes place between the P2 and P3 stem-loops. The core of the aptamer forms the ligand binding site and contains the cytosine (C74) that forms a base pair with the ligand. The secondary structure consensus previously published was revisited using additional aptamer sequences and 3 base positions were identified in the aptamer that display a different sequence requirement. The validity of our observations was assessed by performing a mutational analysis in which 2-aminopurine was used to determine the binding affinity of each variant. We find that one position in the core, which was previously identified as non-conserved and which is exposed to the solvent according to the crystal structure, exhibits a sequence requirement that appears to be related to the ligand specificity of the riboswitch. In addition, the transcriptional control of a natural Gbox variant was studied using real-time PCR. The endogenous ‘on’ and ‘off’ transcript forms of the riboswitch were quantified and the proportion of both was determined in B. subtilis cells grown at different guanine concentrations. This work establishes for the first time that an endogenous guanine riboswitch control gene expression at the transcriptional stage, and we are currently investigating other natural guanine riboswitch variants.
24 . Samuel Dugré-Brisson, George Elvira, Karine Boulay, Laurent Chatel-Chaix & Luc DesGroseillers
Interaction of Staufen1 with the 5' end of mRNA facilitates translation of these RNAs
Département de Biochimie, Université de Montréal, Montréal, Qc, Canada H3C 3J7
Staufen1 is a component of transported ribonucleoprotein complexes. Genetic work in Drosophila has suggested that Staufen plays a role in the derepression of translation of oskar mRNA following localization. To determine whether Staufen1 can play a similar role in mammals, we studied translation of transcripts in the presence or absence of Staufen1. Translationally-repressed mRNAs were generated by fusing the structured HIV-1 transactivating response (TAR) element to the 5’end of a reporter transcript. In rabbit reticulocyte lysates and in mammalian cultured cells, the addition of Staufen1 resulted in the upregulation of reporter activity when translation was driven by the TAR-bearing RNA. In contrast, Staufen1 had no effect on translation of efficiently translated mRNAs lacking an apparent structured 5’end, suggesting that Staufen1-binding to the 5’end is required for enhanced translation. Consistently, Staufen1 RNA-binding activity is necessary for this translational effect. In addition, similar upregulation of translation was observed when Staufen1 was tethered to the 5’end of mRNAs via other structured RNAs, the highest level of translational increase being obtained with the bona fide Staufen1-binding site of the Arf1 transcript. The expression of Staufen1 promoted polysomal loading of TAR-luciferase transcripts resulting in enhanced translation. Our results support a model in which the expression of Stau1 and its interaction with the 5’end of RNA and ribosomes facilitate translation initiation.
25 . Nicolas Paquin, Marie Ménade, Guillaume Poirier, Damiane Donato and Pascal Chartrand
Yeast casein kinase activates the translation of the bud-localized ASH1 mRNA via phosphorylation of the eIF4G-binding protein Khd1p.
Université de Montréal, Department of Biochemistry
In S. cerevisiae, ASH1 mRNA is localized to the bud tip in late anaphase cells. This allows Ash1p, a transcriptional repressor of HO, to be sorted exclusively to the daughter cell nucleus, where it prevents mating type switching. A previous study has reported that overexpression of Khd1p, a three KH domain RNA binding protein, inhibits the translation of ASH1 mRNA. We now report that the disruption of KHD1 leads to a two fold increase in the level of expression of Ash1p. We have found that Khd1p binds the localization element E1, which is present within the coding sequence of ASH1 mRNA. Interestingly, Khd1p interacts in vivo and in vitro with eIF4G1, more specifically with the C-terminal domain of eIF4G1. As this interaction is RNA-dependant, we found that the C-terminal domain of eIF4G1 was also able to bind the localization element E1. Interestingly, we found that the yeast casein kinase Yck1p, a bud-localized kinase, is also involved in the regulation of the expression of Ash1p. Deletion of YCK1 resulted in a decreased expression of Ash1p. Normal expression of Ash1p was recovered in a double knockout of KHD1 and YCK1. Moreover, Yck1p can phosphorylate Khd1p in vitro. We therefore propose that Khd1p acts via a novel translational regulation mechanism by which it regulates the translation of ASH1 mRNA by interacting with the C-terminal domain of eIF4G1, thus affecting the recruitment of ribosomes to this transcript. This translational repression may be released when Yck1p phosphorylates Khd1p as ASH1 mRNA reaches the bud tip.
26 . Valerie S. Greco-Stewart and Martin Pelchat
The RNA polymerase II complex and the nuclear protein PSF bind specifically to the terminal stem-loop domains of both polarities of hepatitis delta virus RNA
University of Ottawa Department of Biochemistry, Microbiology, and Immunology
The hepatitis delta virus is a subsatellite RNA virus of the hepatitis B virus and exacerbates the symptoms of hepatitis. Its genome is comprised of a small, single-stranded RNA molecule with limited protein coding capacity. Remarkably, HDV lacks an RNA-dependent RNA polymerase for its transcription and replication and must coerce host cellular factors to accomplish these processes. In the present study, we confirm the interaction of RNA polymerase II and HDV RNA using both in vitro and in vitro co-immunoprecipitation assays. Interaction of RNAP II with the HDV genome was determined to be confined to the terminal stem-loop domains of both polarities of HDV RNA. Mutations in the conserved 6nt stem of the genomic polarity and the 7nt stem of antigenomic polarity that disrupt the sequence and/or structure of these RNAs abrogated RNAP II binding. Recently, we discovered that the nuclear protein PSF interacts with HDV RNA; in vitro and in vivo co-immunoprecipitation strategies were used to determine the regions of HDV RNA that interact with PSF. It was found that PSF binds the terminal stem-loop domains of HDV as was observed with RNAP II. The binding specificity was verified by EMSA analyses using purified PSF. Preliminary data show that PSF might mediate binding of the RNAP II complex to HDV RNA, suggesting a putative role for these interactions in transcription initiation. Together, these data confirm the involvement of RNAP II in HDV biology and provide insight into the involvement of a novel host protein in the HDV life cycle.
27 . Guillaume Desnoyers, Hubert Salvail, Karine Prévost, Émilie Phaneuf, Jean-François Jacques, Maxime Desmarais, Eric Massé
Activation and repression of two groups of mRNAs by the same small RNA in Escherichia coli
Université de Sherbrooke, Département de Biochimie
Small RNAs (sRNAs) are normally smaller than 300 nucleotides and they usually do not encode for proteins. During the last few years, at least 60 sRNAs have been identified in E. coli and it has been shown that many of them play an important role in translation regulation. One of them, called RyhB, is a sRNA expressed in the absence of iron, via the control of the Fur repressor. It has previously been shown by our group that RyhB causes the degradation of many mRNAs encoding iron-utilizing proteins, and that the RNA chaperone Hfq is required for activity and stability of RyhB. Here we demonstrate that RyhB also activates the translation of at least one mRNA, shiA, encoding a transporter of shikimate, used in the siderophore biosynthesis pathway. This activation occurs via a base-pairing interaction between RyhB and shiA mRNA, that is tought to free the ribosomes-binding site and the translation start codon of shiA mRNA. We have in vivo and in vitro data showing this interaction between RyhB and shiA. Although others sRNAs are known to activate the degradation of target mRNAs, it is the first time that a sRNA is shown to participate in active degradation as well as activating translation of mRNAs.
28 . Shen ZF. , Paquin N. , and Chartrand P.
Exploring the role of the nucleo-cytoplasmic shuttling of She2p in mRNA localization in the budding yeast
Université de Montréal, Department of Biochemistry.
In the yeast Saccharomyces cerevisiae, active transport and localized translation of the ASH1 mRNA at the bud tip is a process that is required for the regulation of mating type switching. She2p is the RNA binding protein involved in the recognition of the cis-acting localization elements present in the ASH1 mRNA. Recently, the 3D structure of She2p has been determined and shown that it acts as a homodimer. This protein actively shuttles between the nucleus and the cytoplasm in an RNA-dependent manner. For now, the function of She2p in the nucleus is still unknown. Our data show that She2p contains an active nuclear localization sequence (NLS) required for its nuclear import. We have evidence that nuclear import is mediated via the importin alpha Srp1p. Interestingly, Srp1p cannot interact with the She2p dimer. However a strong interaction was observed with monomeric She2p in vitro and in vivo, suggesting that a conformational change in this protein is required for its nuclear import. We are currently exploring the role of post-translational modification in the regulation of the She2-Srp1 interaction. We expect that these experiments will shed light on the role of the nucleo-cytoplasmic shuttling of She2p on mRNA localization.
29 . Philippe Cloutier & Benoit Chabot
Charaterization of exonic enhancers and a cryptic 5’ splice site that modulate the production of the anti-apoptotic protein Bcl-xL
Université de Sherbrooke
Sequences within the pre-mRNA often guide splicing choices resulting in a specific set of isoforms. Bcl-x utilizes two competing 5’ splice sites to produce the pro-apoptotic isoform Bcl-xS and the anti-apoptotic variant Bcl-xL. A 83 nt region (B3) is located between these 5’ splice sites and is known to enhance usage of Bcl-xL. Using deletion mutagenesis, we have narrowed down the cis-acting elements within B3. Gel shifts, crosslink and over-expression assays support a role for the RNA-binding protein SRp30c in the activity of B3. In addition, we have identified within B3 another region that has an opposite effect on splicing, thereby impeding the production Bcl-xL. RNA affinity chromatography and RT-PCR assays suggest that this element is bound by snRNP and contains a 5’ splice site that is used when the downstream Bcl-xL 5’ splice site is compromised. We are currently trying to ascertain whether the binding of splicing factors at this site interferes with splicing at adjacent sites.
30 . Guillaume Jannot, Isabelle Banville, Marie-Eve Boisvert and Martin J. Simard
Identification of new components of the microRNA pathway
Centre de Recherche en cancérologie de l’Université Laval, Québec, Québec, Canada
MicroRNAs are required to precisely regulate gene expression in a spatio-temporal manner: an essential feature in many biological processes. Despite the fact that microRNAs are crucial for the animal homeostasis, few aspects of this pathway are known. For uncovering the “nuts and bolts” of this molecular process, our lab focus on the identification and characterization of important proteins associated with the microRNA pathway using C.elegans as a model system. To isolate new players, we are using two approaches: 1) affinity purification of the microRNAs complexes and, 2) yeast two-hybrid screen using the Argonaute protein ALG-1 as a bait (an essential component of the microRNA pathway). Once identified, candidates are knock-down by RNAi and phenotypes are compared with dicer and alg-1 mutants, two genes implicated in the pathway. Candidates that phenocopy the loss-of-function of these genes will be carefully studied in order to demonstrate their implications in the microRNAs pathway. To date, among the candidates identified, one gene isolated by yeast two-hybrid screen displays a loss-of-function phenotype identical to the alg-1 and dcr-1 mutant and thus probably involved in the microRNA pathway. The discovery of important players will lead to a better comprehension of the microRNA pathway.
31 . Evelyne Rondeau and Martin J. Simard
A new genetic screen to uncover microRNA components in C. elegans
Centre de Recherche en Cancérologie de l’Université Laval, Québec, Québec, Canada
MicroRNAs are small non-coding RNAs which have the role of regulating genes by blocking protein synthesis for specific messenger RNA. This cellular mechanism is essential for development and survival of eukaryotes, but few is known about factors involved. We use the nematode Caenorhabditis elegans as a model system to identify, using biochemical and genetic approaches, new cellular factors essential for the metabolic pathway of gene silencing by microRNAs. We designed a genetic screen based on synthetic lethality feature of alg-2 gene: when alg-2 along with another gene, like alg-1, are not functional simultaneously, the animal can not survive. This screen will be performed with a transgenic animal carrying an extrachromosomic array containing the alg-2 gene linked to a broadly expressed GFP gene and in which the genomic copy of alg-2 gene is deleted. According to this, it will be easy to determine, by GFP expression, if the extrachromosomal copy of alg-2 gene is essential for the survival of worms. This worm line is mutagenized with chemical products and candidates that express GFP protein are collected. Using well-developed mapping techniques available in C. elegans, the affected genes in clones recovered will be identified. We believe that this synthetic lethal screen will allow us to identify new genes that work in synergy with alg-2, like alg-1, and this will uncover new components of the microRNA pathway.
32 . Vincent Boissonneault, Denis Soulet, Serge Rivest and Patrick Provost
Regulation of mouse ß-amyloid precursor protein converting enzyme 1 (mBACE1) by miR-298 and miR-328
Centre de Recherche en Rhumatologie et Immunologie and Laboratory of Molecular Endocrinology, Centre de Recherche du CHUL - CHUQ, Department of Anatomy and Physiology, Faculty of Medicine, Université Laval.
MicroRNAs (miRNAs) are ~21- to 23-nucleotide (nt) endogenous RNAs involved in regulating gene expression through repression of messenger RNA (mRNA) translation. Their action is mediated upon recognition of specific binding sites (BS) mainly located in the 3' nontranslated region (NTR) of mRNAs in most eukaryotes. In previous studies, postmortem analysis showed higher ß-amyloid precursor protein converting enzyme (BACE) protein, but not mRNA, levels in the brain of patients suffering from Alzheimer's disease, as compared to normal brains. These observations are suggestive of a possible impairment of miRNA-mediated regulation of BACE expression. We tested this hypothesis by studying the regulatory role of mBACE1 3'NTR in cultured mammalian cells. When inserted downstream of a Renilla luciferase (Rluc) reporter gene, this element reduced Rluc activity by ~70%. Computational analyses revealed the presence of two putative miRNA BS for miR-298 and miR-328. These miRNAs specifically bound to their respective BS in vitro, as assessed by electrophoresis mobility shift assays (EMSA). These observations were confirmed in cultured neuronal cells, in which pre-miRNA overexpression or transfected miRNA duplexes downregulated the expression of Rluc reporter genes coupled to partial or complete 3'NTR of mBACE1. miRNA-regulated Rluc expression was abolished upon introduction in the mBACE1 3'NTR of point mutations disrupting miR-298 and/or miR-328 recognition. Our results support the involvement of miR-298 and miR-328 in mBACE1 gene regulation and offer new perspectives on the etiology of Alzheimer's disease. This study is supported by NARSAD, the Mental Health Research Association.
33 . J-F. Fisette and B. Chabot
hnRNP F, H and A1 : shaping the pre-mRNA using G force
Département de microbiologie et d’infectiologie, Faculté de médecine et des sciences de la santé. Université de Sherbrooke, Sherbrooke, Canada
Binding sites for hnRNP A/B proteins influence the alternative splicing of several pre-mRNAs. We have proposed that bound hnRNP A/B proteins self-interact, thus looping-out and repressing internal splice sites. Using in vitro splicing assays, we have previously showed hnRNP F/H could affect alternative splicing in a manner similar to hnRNP A1. Moreover, experiments including combinations of hnRNP A1 and hnRNP F/H binding sites are also active, suggesting that heterotypic interactions between these proteins are permitted. Using hybrid proteins, we observed that hnRNP F/H and hnRNP A1 require similar protein domains for their effect on splicing. Glycine rich domains (GRD) are a key determinant: the splicing activity of hnRNP H was lost when we removed this domain and was restored when the GRD of hnRNP A1 was added. Also, we have investigated the potential role of hnRNP F/H in the alternative splicing of exon 7B from the A1 pre-mRNA. We observe that the knockdown of hnRNP H stimulates inclusion of this exon. We found by gel shift assay that hnRNP H could bind to a G-rich sequence located in exon 7B. Using heterologous system, we would like to confirm the contribution of this hnRNP H binding site and address its mechanism of action. Specifically, we would like to assess whether a hnRNP H bound to this exonic element can interact with A1 molecules bound in flanking introns. The existence of such a network of heterotypic interactions would indicate that interactions between different hnRNP proteins represent a novel mechanism of splicing control.
34 . Timothée Revil and Benoit Chabot
Analysis of two elements and a pathway in bcl-x splicing regulation
Département de microbiologie et d’infectiologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada
Alternative splicing of bcl-x can lead to two major isoforms with antagonistic activities that can modulate cell death or survival. We are investigating the factors and elements that are implicated in this important splicing decision. Using deletion mutagenesis, we have identified two exonic elements situated upstream of the 5’ splice site of the pro-apoptotic isoform, bcl-xS. The first element, B1, contains two distinct sub-regions with antagonistic activities. hnRNP K interacts with a portion of the B1 element in RNA chromatography assays. Knocking down hnRNP K with RNAi affects bcl-x splicing in PC-3 cells in a way that suggests that it may be competing with another factor for the binding on B1. Additional experiments are being performed to assess the molecular mechanisms by which B1 affects bcl-x splicing. Another element, SB1, is responsible for a staurosporine-mediated shift towards the pro-apoptotic bcl-xS in 293 cells. Analysis of this element indicated that this element normally inhibits downstream splice site utilization until inhibition of protein kinase C using staurosporine or other specific inhibitors relieves this repression. The SB1 element can act in a heterologous pre-mRNA. We are currently investigating the factors and specific sequences implicated in this pathway.
35 . Gabriel Lapointe and Luc DesGroseillers
Characterization of Staufen 2 containing complex in neurons.
Département de Biochimie, Faculté de Médecine de l'Université de Montréal
Neuronal activity depends on information exchange between different neurons. To achieve this task, neurons need to produce synapses far away from the cell body. These synapses not only contain all the necessary receptors and neurotransmitters, but they must also be complementary to the target cell. It is now understood that mRNA coding for these proteins are transported along the dendrites and axons to be translated locally. Staufen 2 is a double stranded RNA binding protein highly expressed in the brain. It is also an integral part of the dendritic RNA transport granules and is essential for mRNA transport. We identified several Staufen 2 interacting proteins using the yeast double hybrid system. Among these are the ribosomal protein S7, makorin 1, áB-crystallin, rpt2, DSCR1 and WHSC1.These proteins are either known to be an essential part of synaptic regulation or implicated in diseases. Our goal is to characterize these interactions and to evaluate their physiological relevance.
36 . Alex Beribisky, Mina Motamed, Sharon Zhao, Tony Tavares and Philip. E. Johnson
Determination of the three-dimensional structure of the Selenocysteine Insertion Sequence RNA from Moorella thermoacetica using Nuclear Magnetic Resonance Spectroscopy
Department of Chemistry, York University, Toronto, Ontario, Canada
The messenger RNA (mRNA) codon UGA normally serves as a signal for termination of protein synthesis. However, this codon can also promote the incorporation of the non-standard amino acid selenocysteine (Sec). The main conditions for Sec incorporation are the presence of a mRNA hairpin called the Selenocysteine Insertion Sequence (SECIS) at a certain distance from the UGA codon which serves as the binding target for a protein called SelB. While the process of Sec incorporation is essentially the same in all prokaryotes, the SECIS structures vary quite bit among these organisms. The E. coli SECIS contains a bulged uracil residue within its stem while M. thermoacetica SECIS lacks one and each of these two RNAs has its own distinct loop sequence and structure. There is also a large variation in binding affinity of the SelB proteins to their cognate RNAs, as the E. coli SelB binds a thousand times tighter than its M. thermoacetica counterpart. Recent work on the M. thermoacetica SECIS / SelB complex has shown that SECIS RNA from this organism binds its own SelB in a unique manner. However the structure of the free SECIS RNA remains to be elucidated. Our research involves the determination of the three-dimensional structure of the unbound M. thermoacetica SECIS RNA hairpin using NMR methods. A SECIS RNA sample was prepared and 2D homonuclear as well 3D hetronuclear NMR spectral data was obtained. The resonances are assigned and progress in determining the structure will be presented.
37 . David Chiovitti and Philip E. Johnson
Design of a RNA Affinity Tag for the Purification of RNA Molecules from Bacteria
Department of Chemistry, York University
Isotopically labelled RNA molecules are important for obtaining high resolution structural data using NMR spectroscopy. However, it is expensive to make labelled RNA using conventional in vitro transcription methods. We aim to design an efficient method of producing labelled RNA biosynthetically in bacteria grown in labelled media. This goal necessitates an efficient purification method, we are developing a chromatography based purification scheme modified from the recently introduced method of Kieft and Batey (RNA (2004), 10:988-995) to purify RNA molecules from E coli cells. Progress towards the production of labelled RNA will be presented.
38 . Roman Rydzanicz
Structural studies of RNA segments of tomato bushy stunt virus using NMR
York University
Tombusviridae is a large positive-strand RNA virus family. Positive-strand RNA virus genomes are copied into negative-sense RNA complements that are then used as templates for the production of progeny genomes. Members of this family have RNA genomes that terminate with CCC-OH. Tomato bushy stunt virus (TBSV), the prototype virus of this family, has a genome ending with AGCCC-OH, termed the 3' complementary silencer sequence (3'CSS). The 3'CSS is able to base pair with a complementary internally-located sequence, 5'GGGCU, called the replication silencer element (RSE). In TBSV, previous compensatory mutational analysis of the RSE-3'CSS interaction showed it to be functionally important for viral RNA synthesis both in vitro and in vivo. Data suggest that accessibility of the elements and their proximity to adjoining stem structures are important functional parameters. Furthermore, a working structural model for the TBSV RSE-3'CSS interaction has been proposed that involves coaxial stacking of adjacent helices at either end of the RSE-3'CSS interaction. Preliminary computational and NMR structural results will be presented.
39 . Dorota Sikora and Martin Pelchat
Identification of an ASF/SF2 binding site on the hepatitis delta virus RNA
Department of Biochemistry, Microbiology and Immunology. Faculty of Medicine. University of Ottawa, Ottawa, Ontario, Canada
The hepatitis delta virus (HDV) is the only subviral RNA pathogen known to infect humans. In natural infections, HDV is closely associated with the hepatitis B virus (HBV), on which it relies for transmission. Its small genome (~1700 nt) consists of a single stranded, circular RNA molecule. HDV encodes two proteins, the large and small hepatitis delta antigens. Because HDV cannot replicate its own genome, it is hypothesized that it takes advantage of numerous host proteins to ensure its propagation. We used in vitro binding assays, such as the electrophoretic mobility shift assay, and co-immunoprecipitation analyses to screen a number of potential HDV binding proteins. We have identified the human splicing factor ASF/SF2 as a binding partner for HDV RNA. Using fluorescence experiments, we were able to locate an ASF/SF2 binding site. Our results will be invaluable in further research into the mechanisms by which HDV makes use of the host cell's proteins to direct its own replication.
40 . Mina Motamed, Philip Johnson
Structural Study of Cocaine Aptamer
Department of Chemistry York University Toronto ON, Canada
A DNA based cocaine aptamer has been previously developed that has a strong affinity for cocaine. Cocaine binds to the aptamer via a hydrophobic pocket formed by a non-canonical three-way junction. The unbound aptamer has a loose structure with two open stems. However, when cocaine is present, the molecule folds to form the three-way junction structure. Considering this major structural change, we are studying the structure of the aptamer both in its free and bond form using NMR methods, preliminary results will be presented.
41 . Marie-Eve Boisvert, Isabelle Banville and Martin J. Simard
Playing with the small RNA pathways
Centre de Recherche en Cancérologie de l'Université Laval, Québec, Québec, Canada
In C. elegans, the molecular characteristics the siRNA and the microRNA pathways are different. In the siRNA pathway, a perfectly based-paired dsRNA molecule initiates the targeted mRNA degradation. In contrast, the stem-looped microRNA contains an imperfect base-pairing, leading to translational inhibition of the mRNA. Also, each of these pathways has its own argonaute proteins. The siRNA pathway requires RDE-1, instead of ALG-1 and ALG-2 which act in the microRNA pathway. One reason of this disparity could be that trigger molecules are recognized by specific factors that “imprint” the RNA molecule and guide to the appropriate outcome. To better understand the differences between the siRNA and the microRNA pathways, we designed an siRNA containing a microRNA sequence and a microRNA targeting the GFP gene. To date, in a let-7 depleted strain, we can see a rescuing of the let-7 phenotype when the worm is injected with a let-7 siRNA, and this rescue seems dependent of some siRNA factors. Our recent data will be discuss at the meeting.
42 . De Guire V*., Sylvestre Y*., Querido E., Mukhopadhyay U. K., Chartrand P., Ferbeyre G. (*co-author)
A self-regulatory pathway between miR-20 and E2F transcription factors.
Département de Biochimie, Université de Montréal, Montréal, QC H3C 3J7, Canada.
More than 50 % of miRNAs genes are amplified, deleted or translocated in tumors. Our research program is based on the study of the miR-17-92 cluster that contains several homologous miRNAs. The genomic locus of the cluster is amplified in B cell lymphoma, follicular lymphoma and other tumor types, suggesting that it has oncogenic properties. Based on bioinformatics analysis, we investigated whether that miR-20, one of the miRNAs of the cluster, targets the mRNA of E2F1, E2F2 and E2F3, a family of protein that has an essential role in cell progression and apoptosis. To do so, we fused the 3’UTR of the E2Fs mRNA to a luciferase reporter. When we used an antisense RNA against miR-20 or mutated the binding sites of this miRNA in the 3’UTR the E2Fs, we observed an increase in the luciferase fluorescence in HeLa cells. Using bioinformatics analysis, we identified several potential binding sites for the E2Fs transcription factors in the promoter of miR-17-92. We were able to observe an increase in miR-20 levels in HeLa cells overexpressing members of the E2F family, which shows the capacity of these transcription factors to activate the transcription of the cluster. We also have biology data proposing that the oncogenic propreties of miR-17-92 cluster could be trough the decrease of apoptosis that is dependant of E2F1. These results suggest the existence of a negative feedback mechanism between miR-20 and the E2F family.
43 . Nadeem Siddiqui, David Mangus, Tsung-Cheng Chang, Jeanne-Marie Palermino, Ann-Bin Shyu and Kalle Gehring
The role of the PABC domain in mRNA metabolism
Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0122, USA.
The poly-(A) binding protein (PABP) is an essential RNA binding protein found in all eukaryotes and is involved in an extensive range of cellular functions including translation, mRNA metabolism, and RNA export. Its C-terminal region contains a peptide interacting ‘PABC’ domain which recruits proteins containing a highly specific ‘PAM-2’ sequence motif to the mRNP complex. In humans, these proteins, including paip1, paip2, eukaryotic release factor 3, ataxin-2 and tob2, are all found to regulate translation in varying ways. We report poly (A)-nuclease (PAN) as a PABC interacting partner in both yeast and humans. The interaction is mediated by a PAM-2 motif identified in PAN. This site can be identified in various fungi and metazoans indicating that the interaction is conserved throughout evolution. These results implicate a novel role for the PABC domain in mRNA metabolic processes and give further insight into PABP’s function in mRNA biogenesis, export and turnover.
44 . Mélina Arguin, Karine Prévost and Eric Massé
Hfq RNA chaperone predetermines mRNAs targeted by a small regulatory RNA
Département de Biochimie, Faculté de Médecine et des Sciences de la santé, Université de Sherbrooke
Interest has raised for small RNAs (sRNA) since they have been shown to be important metabolic regulators and stress adaptors in bacteria. In Escherichia coli, there are more than 60 known sRNAs, which are non coding for protein and are usually smaller than 300 nucleotides. One of those sRNAs, RyhB, is implicated in iron metabolism. The regulation of ryhB gene is under the control of the iron-sensing Fur repressor, and is expressed during iron starvation. Once expressed, RyhB finds its mRNA targets (20) by partial basepairing, then the RNA duplex is degraded via RNase E. Because all RyhB mRNA targets encodes for iron-using proteins, this mechanism allows tight regulation of iron utilization when the metal is scarce. Little is known about the pairing and the specificity of RyhB with its mRNA targets. However, it is well known that Hfq, an RNA chaperone, is essential for RyhB activity and is thought to promote the pairing by binding to each RNA, bringing them to close proximity. Here we characterized the binding of Hfq on four RyhB mRNA targets and two non-targets. Our in vitro data show that Hfq binds more strongly to the RyhB mRNA targets as compared to non-targets. Additionally, in ternary complexes assays, we noticed that pairing between RyhB and its mRNA targets was largely enhanced by Hfq, and that Hfq was ineffective for non-targets. Finally, we propose that Hfq binding on mRNAs helps determine the specifity of RyhB targets recognition.
45 . Moheshwarnath Issur and Martin Bisaillon
Investigating into the GTP specificity of viral RNA guanylyltransferase by using the PBCV-1 capping enzyme as model
Université de Sherbrooke
Various viruses of clinical significance possess enzymes involved in the formation of a cap structure at the 5’ end of their mRNAs. The cap structure, a 7-methylguanosine residue linked by a 5’-5’ triphosphate bridge to the RNA transcript, is of fundamental importance to the stability, half-life and transport of mRNAs. The synthesis of this cap structure is carried out by three sequential enzymatic reactions, the first one involving the hydrolysis of the RNA 5’ triphosphate terminus by an RNA triphosphatase to form a diphosphate end, the second step being the transfer of a GMP to the diphosphate end of the RNA by an RNA guanylyltransferase, which uses GTP as a substrate, and finally followed by the methylation of the GpppN cap by an RNA (guanine-N7) methyl transferase. Despite several similarities with cellular homologs, viral-encoded capping enzymes also present significant differences and as a result, are potential targets for the development of anti-viral drugs. The aim of this research is to investigate into the GTP specificity of the RNA guanylyltransferase, by using various purine triphosphate analogs as substrates for the PBCV-1 RNA guanylyltransferase (the smallest known enzyme of this family). This will also give an insight into the viral capping mechanism, hereby enabling precise targeting for the elaboration of anti-viral drugs.

Poster Session 2

46 . Cédric Reymond, Jonathan Ouellet, Martin Bisaillon and Jean-Pierre Perreault
Localization of the 5’-end of the delta ribozyme
Université de Sherbrooke
The comprehension of RNA folding and the kinetics forces behind it, is probably the main difficulty when we want to understand RNA behaviour. Delta ribozyme and ribozymes in general, because they associate a function with a structure, are extremely valuable tools for this kind of study. Along its folding pathway, the delta ribozyme can adopt a huge variety of different structures including kinetic traps and misfolded conformations. Some of these intermediates can be analysed by crystallography or nuclear magnetic resonance but these methods only give three dimensions structures of stable intermediates, furthermore most did not localize the 5'-end of the ribozyme. In this work we want to ask this question because we believe that it is a major issue to explain the catalytic activity of this ribozyme. Cross-linking experiments have been made on the trans-acting antigenomic delta ribozyme coupled with specific mutations in the ribozyme and/or the substrate to establish the localisation of the 5'-end of the substrate. Moreover, we have performed a steric study of the catalytic core with substrates designed for that purpose and enzymatic probing of the position -1 to -3 with RNase H and specific DNA probes. Our current results are confirming unpublished data from our lab observations of the 5'-end entrapped inside the ribozyme by the formation of the P1.1 stem.
47 . Kim Marsh and Alan Cochrane
Mechanism of Sam68DC inhibition of HIV-1
Department of Medical Genetics and Microbiology, University of Toronto
Sam68DC is a potent, specific inhibitor of HIV-1. Sam68DC bundles the HIV-1 mRNA at the nuclear periphery, and we hypothesized that this made the mRNA inaccessible to polysomes. We were able to show that the bundling was dependent on intact actin microfilaments. However, upon disocciation of the microfilaments, and the Sam68DC-mRNA bundles, there was still no translation. Therefore, Sam68DC acts specifically to inhibit the translation of the unspliced, and incompletely spliced classes of HIV-1 mRNA. We have shown thaat the mRNA still has an intact 5'cap and 3'polyA tail in the presence of Sam68DC. Our recent work has elucidated differences in composition of the mRNP in the presence of Sam68DC.
48 . Carlos Melendez-Peña, Sébastien Lainé, Anne Gatignol.
Novel assays to test RNAi function and viral suppressors of RNAi.
Lady Davis Institute For Medical Research, Department of Microbiology and Immunology, McGill University, Montréal, Qc, Canada.
RNA silencing is postulated to be an ancient immune mechanism used by cells to restrict viruses, transgenes, and transposons. Consistent with this notion, RNA interference (RNAi) is a natural antiviral mechanism in plants and insect cells and it can also act against viruses in worms. The human immunodeficiency virus HIV-1 TAR RNA binding protein (TRBP) interacts with Dicer in mammalian cells and is a component of the RNA-induced silencing complex. TRBP is a double stranded RNA binding protein that increases translation from human immunodeficiency virus TAR containing RNAs by a direct binding to the structured TAR and inhibiting the interferon-induced protein kinase PKR. Our aim is to better understand the interaction between Dicer and TRBP, TRBP’s requirement in RNAi and the activity of RNAi suppressors from mammalian viruses. Using the two-hybrid method we identified the specific region of interaction in the C-terminal part of TRBP (C3) and in the N-terminal part of Dicer (D1). We developed a FACS assay based on fluorescence inhibition by short-hairpin and micro RNAs against GFP. We showed that RNAi is active in HeLa cells but not in "knocked-out" Tarbp2 -/- mouse cells. A dual luciferase assay showed that E3L of the Vaccinia virus and NS1 of Influenza virus are also active suppressors of RNAi in mammalian cells, as well as HIV-1 Tat protein and we currently test the HIV-1 TAR RNA.
49 . Viranaicken W., Larcher J.C., Gasmi L., Bernard R., Eddé B. and Denoulet P.
Interleukin enhancer binding factor 3 (Ilf3) and Nuclear factor 90 interacts with the axonal targeting element of Tau mRNA
Faculté de Médecine, Département de Biochimie, Université de Montréal, QC, Montréal H3JT4 Biochimie Cellulaire, UMR 7098, CNRS - Université Paris 6, 75252 Paris Cedex 05, France Neurobiologie des signaux intercellulaires, UMR 7101, CNRS - Université Paris 6, 75252 Paris Cedex 05, France Centre de Recherches de Biochimie Macromoléculaire, UPR 1086, CNRS, 34293 Montpellier Cedex, France
In developing organisms (Drosophila, Xenopus, ?) or in differentiated cells (neurons, fibroblasts, ?), many mRNA are asymmetrically localized in cytoplasm. These selective localizations are due to the presence of specific sequences and/or particular structural motifs in mRNA to be transported (cis-acting element) that interact with trans-acting proteins. In neurons, some mRNA are delivered to specific subcellular compartments, notably Tau and MAP2 localized in axons and dendrites, respectively. The particular distributions of Tau and MAP2 proteins, favored by the prelocalization of their mRNA may explain neuronal asymmetry. It has previously been shown that the selective translocation of Tau mRNA in axons relies on the integrity of microtubules (Litman et al., 1996, Neuron, 13, 1463-1474) and is due to a 91 nucleotide-segment present in the 3'-untranslated region of Tau mRNA (Behar et al., 1995, Int J Dev Neurosci, 13, 113-127), serving as RNA axonal targeting element (ATE). Using this ATE as radioactive probe in a North-Western approach, we have identified some ATE binding proteins that interact with the targeting RNA element. Two proteins are identify, Ilf3 (Interleukin enhancer binding factor 3) and NF90 (Nuclear factor 90). Different approach are use to determined involvement of these two factors in mRNA localization.
50 . Charbonneau, J., Gendron, K. ,Heveker, N. , Ferbeyre, G & Brakier-Gingras, L.
Influence of translation initiation on the efficiency of a ribosomal frameshift in the human immunodeficiency virus type 1 (HIV-1)
Département de biochimie, U. de Montréal et Centre de Recherche, Hôpital Sainte-Justine
HIV-1 uses a programmed –1 ribosomal frameshift during translation of its full-length mRNA to produce Pol, the precursor of its enzymes. This abnormal event is critical for viral infectivity. We investigated whether changes in the translation initiation rate influence the frameshift efficiency. We used bicistronic vectors where the HIV-1 frameshift region is inserted between the Renilla and firefly luciferase genes. Only ribosomes that make a frameshift produce the firefly luciferase. We inserted the TAR stem-loop (trans-activator response region), which is present at the 5’ end of all HIV-1 mRNAs, upstream the Renilla coding sequence to mimic the situation encountered by ribosomes translating viral mRNAs in infected cells. We transfected Jurkat cells with these vectors to assess the frameshift efficiency. TAR can activate PKR (RNA-activated protein kinase), depending upon the conditions, and, consequently, decrease the rate of translation initiation, which was related to an increase in the level of phosphorylation of the initiation factor eIF2. TAR contains binding sites for various cellular and viral proteins, such as the viral protein Tat. When translation initiation is cap-dependent, we observed that TAR increases the frameshift efficiency and this indicates that the frameshift efficiency is influenced by changes in translation initiation. We also observed that the presence of Tat counteracts the effect of TAR. Finally, preliminary results led us to suggest that HIV-1 uses an IRES (cap-independent entry site) for the translation of its full-length mRNA, which avoids variations in frameshift efficiency that could occur with a cap-dependent initiation of translation.
51 . Alexey Yu. Denisov, Guennadi Kozlov, Michel Gravel, Peter E. Braun, Kalle Gehring
Solution structure of regeneration-induced CNPase homolog (RICH)
Department of Biochemistry, McGill University, Montreal, H3G 1Y6
RICH protein is a 2',3'-cyclic-nucleotide 3'-phosphodiesterase induced during optic nerve regeneration in fish. The protein contains an N-terminal domain rich in negatively-charged residues and a catalytic C-terminal domain, which possesses 35% sequence homology to the catalytic domain of mammalian brain 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). Here, we use nuclear magnetic resonance (NMR) techniques to study the 24 kDa catalytic domain of goldfish RICH. Complete backbone and side chain assignments were obtained and the solution structure of the RICH catalytic domain was determined, which is highly similar to that of the rat CNPase catalytic domain. NMR titrations of this domain with different inhibitors identified specific residues involved in catalytic activity of RICH. These studies may provide a better understanding of the molecular basis of nerve regeneration and to help identify physiological substrates of CNPases.
52 . Roscoe Klinck, Panagiotis Prinos, Benoit Chabot, Sherif Abou Elela
LISA: An integrated system for tissue-specific alternative splicing annotation
Centre de génomique fonctionnelle de l'Université de Sherbrooke
While it is now generally accepted that the majority of human genes undergo alternative splicing (AS), access to experimentally validated tissue-specific annotation of these AS events has been difficult. To address this, we have developed LISA, (Layered and Integrated system for Splicing Annotation), an informatic and experimental platform for the RT-PCR based annotation of AS in human tissues and cell lines. The system comprises in silico primer and experimental design, automated high throughput execution of PCR reactions on total human cDNA, capillary electrophoretic quantitation of amplicons and automated data analysis and filtering modules. The LISA uses publicly available mRNA and EST-based transcript compilations as a starting point for the annotation process. All proposed events are classified by type and their existence and relative abundance is assessed in each RNA source. The system also allows the facile detection of novel AS events. We have tested the LISA on 86 apoptosis associated genes in 6 human RNA sources. 89% of the 1025 AS events predicted for these genes could be validated in at least one of the RNA sources analyzed. One quarter of the genes showed strong evidence for novel AS events, 10 of these were confirmed by sequencing. An in silico data filter was developed and applied to our database to identify AS-associated differences between tumor and normal tissues. For the 86 gene set, this filter yielded an 11 gene subset harboring key AS events which were subsequently evaluated for their functional relevance and potential as candidate AS-based cancer markers.
53 . Léger, M., Boudreau, I., Steinberg, S. & Brakier-Gingras, L.
A programmed -1 ribosomal frameshift occurs during the translocation step of the elongation cycle.
Département de Biochimie Université de Montréal
A programmed -1 frameshift is used to synthesize HIV-1 enzymes when the full-length viral mRNA is translated by the ribosomes of the infected cells. This frameshift occurs at a slippery sequence, a heptanucleotide, followed by a stimulatory signal, a peculiar RNA motif, which promotes frameshifting. Our aim is to understand the mechanism that accounts for HIV-1 frameshift. We first demonstrated that the slippery sequence is longer than a heptanucleotide and is extended by two nucleotides in the 5’ direction. We had previously demonstrated that HIV-1 frameshift can be recapitulated in bacteria. Using a specialized bacterial ribosome system, we introduced random mutations in a plasmid-encoded 16S ribosomal RNA (rRNA). The effect of these mutations on the HIV-1 programmed -1 ribosomal frameshift was studied by measuring the expression of the green fluorescent protein (GFP), in which the HIV-1 frameshift region was inserted in the beginning of its coding sequence. The mutations that were selected are located in helices 21 and 22 of the 16S rRNA, a region involved in conformational changes during the translocation of the tRNAs following peptide bond formation. From these results, we suggest that programmed -1 ribosomal frameshift occurs following an abnormality in the translocational step during the elongation cycle.
54 . Ulrike Froehlich, Eric Paquet, Elvy Lapointe, Sonia Couture, Panagiotis Prinos, Sherif Abou-Elela and Benoit Chabot
Regulation of the Alternative Splicing of Apoptotic genes by hnRNP Proteins
Centre de Génomique Fonctionnelle de l'Université de Sherbrooke, Centre de développement des biotechnologies (CDB) de Sherbrooke, Sherbrooke, Québec, J1E 4K8 Canada Département de microbiologie et d'infectiologie, Faculté de médicine et des sciences de la santé, Université de Sherbrooke, Québec, Canada
Regulation of alternative splicing is mediated to a large extent by RNA-binding proteins. The majority of the genes encoding the more than 200 pro- and anti-apoptotic proteins involved in apoptotic control are alternatively spliced. Regulatory proteins of opposing functions are often produced from a common pre-mRNA. Thus, alternative splicing of apoptotic genes is of a crucial importance for apoptotic regulation. Very little is known about the molecular mechanisms regulating splice site selection of apoptotic genes. We have undertaken a large-scale systematic analysis of the effects of RBP protein knock-down on the alternative splicing of a panel of human apoptotic genes. Analysis of the data supports an intricate regulatory RBP network of apoptotic splicing units.
55 . Théberge-Julien, G., Dulude, D., Ferbeyre, G., Heveker, N, Brakier-Gingras, L.
Selection of peptides that interfere with a programmed -1 ribosomal frameshift in HIV-1.
Département de Biochimie, U. de Montréal et Centre de recherche, Hôpital Ste-Justine.
The precursor of the viral enzymes of HIV-1, Pol, is produced via a programmed -1 ribosomal frameshift. This frameshift occurs at a specific region containing two cis-acting elements: a slippery sequence where the frameshift occurs and a stimulatory signal. This signal is a peculiar secondary structure downstream of the slippery sequence that controls frameshift efficiency. Our objective is to select peptides that can bind to the stimulatory signal of HIV-1 (group M, subtype B) and alter frameshift efficiency, thus harming viral replication. The peptides are obtained from a combinatorial library producing fourteen amino-acid long peptides composed of arginine, histidine, serine and asparagine residues, with a bias for arginine. A dual reporter system was used to screen for efficient peptides. In that system, a red fluorescent protein (RFP) is synthesized by conventional translation whereas only ribosomes that make a -1 frameshift produce a green fluorescent protein (GFP), the gene expressing this protein having the HIV-1 frameshift region cloned at the beginning of the coding sequence. A screen by fluorescence-activated cell sorting (FACS) of E. coli Top10 bacteria transformed with the reporter plasmid and the peptide library allowed us to select about 600 different clones for which the frameshift efficiency was altered. We confirmed that about 20 of these clones significantly alter the frameshift efficiency of the HIV frameshift region. These potentially interesting clones are now investigated for their specificity for the HIV-1 subtype B stimulatory signal and could lead to the characterization of novel anti-HIV drugs.
56 . James Uniacke and William Zerges
Chloroplast mRNAs and ribosomes localize to regions within the posterior lobe for photosystem II biogenesis
Concordia University
Several lines of evidence will be presented that photosystem II biogenesis is initiated in specific regions within the posterior lobe of the chloroplast. For example, chloroplast mRNAs encoding PSII subunits and chloroplast ribosomes rapidly localize near the pyrenoid during the induction of PSII subunit synthesis, as revealed by visualization of signals from immunofluorescence and fluorescence in situ hybridization by confocal microscopy. This localization of the psbA mRNA is independent of its translation, and thus does not require a co-translational protein targeting pathway. Results of inhibition studies suggest that the psbA mRNA is targeted by light-regulated RNA-binding proteins that we have characterized previously. These results have many implications for models of the mechanisms and spatial organization of thylakoid biogenesis.
57 . Michaela Müller, Fréderic Bringaud, Annie Rochette, François McNicoll & Barbara Papadopoulou
Infectious Diseases Research Center, CHUL Research Center, Department of Medical Biology, Laval University, Quebec, Canada (418) 654-2705 [email protected]
Leishmania are unicellular parasites that exist in two developmental stages: free-living promastigotes in the alimentary tract of a sandfly vector and intracellular amastigotes residing in phagolysosomes of mammalian macrophages. Leishmania encounters drastic environmental changes during developmental switches from promastigotes to amastigotes. These developmental stages display distinct morphologic and metabolic characteristics, consistent with a highly regulated level of differential protein expression, which is central to parasite’s intracellular survival. In Leishmania differential stage-regulated gene expression is often mediated by sequences within 3’untranslated regions (3’UTRs). Using in-silico screening and bioinformatic analyses, we recently identified ~2000 short non-LTR retroelements that can be grouped into two distinct classes (I & II). These elements are inserted mainly in intergenic regions between protein coding genes. Interestingly, while investigating the regulatory potential of these elements, we found a large number of class I retroelements inserted in 3’UTRs of developmentally regulated transcripts encoding amastin surface proteins. Using microarray analyses, reporter gene assays and polysome profiling studies we showed that retroelements of class I are associated with stage-specific gene regulation and act as stimulators of translation in Leishmania amastigotes by facilitating the binding of mRNAs to highly translated polyribosomes in response to heat shock. Class II retroelements are also associated with stage-specific regulation, however, their deletion increases luciferase reporter gene expression specifically in amastigotes, which suggests that these elements may act as translational repressors. We are currently investigating the mode of action of these widespread retroelements and the underlying molecular mechanisms that govern developmental gene regulation in this important pathogen.
58 . Laetitia Davidovic, Xavier Jaglin, Maud Gravel, Barthelemy Tournier, Sandra Tremblay and Edouard W. Khandjian
A link between traveling RNP-Granules and the Fragile X Mental Retardation protein: a new neuronal structure
Unité de Recherche en Génétique Humaine et Moléculaire, Centre de recherche Hôpital Saint-François d’Assise, le CHUQ, Québec, (Qc) G1L 3L5 and Département de biologie médicale, Faculté de médecine, Université Laval, Québec, Canada.
The Fragile X Mental Retardation protein (FMRP) is though to be a key player in the control of translation and its absence is the cause of the fragile-X syndrome, the most common inherited form of mental retardation. FMRP is an RNA-binding protein present in messenger ribonucleoprotein (mRNP) complexes associated with the translation machinery; however, the exact role of FMRP in translation remains unclear. While the great majority of FMRP is observed in the neuron cell body in association with polyribosomes, a small fraction is present in RNPs-granule-like structures inferred to contain repressed mRNAs. These complexes are transported to the distant dendrites, and synapses where local synthesis of neuro-specific proteins is taking place. In search for proteins interacting with FMRP, we have picked-up a member of the kinesine family. This allowed us to isolate and purify a specific population of RNP-granules that are associated with microtubules. Evidence will be presented that in addition to target RNA, FMRP acts also as a molecular protein-protein adapter between the RNP granules and motors necessary to translocate RNP-granules. from the cell body to distal locations.
59 . Gravel Maud, Davidovic Laetitia, Tremblay Sandra, and Khandjian Edouard W.
Unité de Recherche en Génétique Humaine et Moléculaire, Centre de recherche Hôpital Saint-François d'Assise, le CHUQ, Québec, (Qc) G1L 3L5 and Département de biologie médicale, Faculté de médecine, Université Laval, Québec, Canada
Inherited mental retardation associated with fragile X syndrome is caused by the absence of the Fragile X Mental Retardation Protein (FMRP). This RNA-binding protein is present in mRNP complexes and possesses nucleic acid chaperone properties. We have presented a model in which FMRP contributes to maintaining mRNP complexes in a repressed state in neurons during their transport from the cell body to distant sites such as synapses where highly specialised translation occurs. In mouse brain the very great majority of FMRP is associated with mRNPs present with heavily sedimenting polyribosomes. In addition, a minor fraction is found associated with travelling granules. We observe two distinct populations of polyribosomal mRNPs containing FMRP; the first is associated with membrane-specific polyribosomes and the second population corresponds to free polyribosomes. The membrane fraction contains rough endoplasmic reticulum and also synaptosomes. Since synaptic development and maturation is dependant on local protein synthesis, we further isolated polyribosomes from synaptosomes and showed that FMRP is also associated with these specialized local structures. The most relevant function of FMRP perhaps resides in its presence at synapses and its association therein with the translation machinery. Work is in progress to characterise mRNAs that are associated with free, membrane bound and synapse polyribosomes that could be translationaly regulated by FMRP, and to study whether neuronal distribution of these RNAs is altered in Fmr1 KO mouse brain.
60 . Chow, Conan; Dumas, Carole and Papadopoulou, Barbara.
The effects of endoplasmic reticulum stress on regulation of global mRNA translation and intracellular survival of Leishmania
Infectious Disease Research Centre, CHUL Research Centre, Department of medical biology, Faculty of medicine, Laval University, Quebec, Canada
Leishmania protozoan parasites have a life-cycle which alternates between motile promastigotes in the sandfly vector and intracellular amastigotes in mammalian macrophages. During its life-cycle, Leishmania experiences drastic environmental stresses that affect intracellular survival of the parasite and this is being exploited to identify new targets for developing drugs against these parasites. In other eukaryotes, the stress response predominantly involves the phosphorylation of the translation initiation factor 2á subunit (eIF2á) by eIF2á kinases that decreases overall translation within the cell. In Leishmania, eIF2á phosphorylation and downregulation of global translation arises when promastigotes become amastigotes and exposure of parasites to heat shock or acidic pH or thapsigargin which induces endoplasmic reticulum (ER) stress. The PERK eIF2á kinase is the principal mediator of ER stress which is caused by the accumulation of unfolded proteins. In silico screening of the Leishmania genome indicates the existence of a single copy putative PERK homologue. The PERK gene is expressed as a ~130 kDA protein, is developmentally regulated in amastigotes and is induced upon thapsigargin treatment suggesting that the regulation of PERK expression in Leishmania may also occur at the posttranscriptional level in contrast to other eukaryotes in which PERK is regulated posttranslationally. Immunofluorescence localisation studies suggest that the Leishmania PERK homologue colocalises with the ER chaperone, BiP, which is known to bind PERK in other eukaryotes. Disruption of one PERK allele results in negligible differences in promastigote growth, however, it markedly retards amastigote growth, which highlights the importance of PERK in the intracellular survival of Leishmania.
61 . G. Desjardins, P. Lampron and P. Legault
Structural studies of the catalytic domain of the Neurospora VS ribozyme
Université de Montréal
We are studying the catalytic domain of the Neurospora VS ribozyme as a model system to increase our knowledge of the structure-function relationship in RNA and to better understand the mechanism involved in the cleavage reaction. A global model of the structure has been determined through FRET experiences, but no crystallographic or NMR structure of the active ribozyme is available at the moment. This model suggests that the stem-loop I docks in the cleft formed by stem-loop II and VI. According to this model and mutagenesis studies, the active site of the VS ribozyme is located in the A730 loop of stem-loop VI (SL6). The adenines in this loop play a critical role in the reaction of the cleavage mechanism. Using NMR spectroscopy, we are currently characterizing the catalytic domain and a fragment containing the active site (SL6). We are using 1D 1H, 2D 1H-15N HSQC and NOESY spectra to structurally characterize the SL6 RNA and the structural data obtained will be compared with those of the catalytic domain. Using C-13 NMR we will determine if an adenine with a shifted pKa is present in the active site of the fragment and in the catalytic domain. These studies will increase our understanding of the mechanism involved in the cleavage reaction by the catalytic domain and it will help us develop a better structural model for this ribozyme.
62 . Bruno Lamontagne and Sherif Abou Elela
RNA-mediated cleavage in vivo and in vitro using eukaryotic RNase III
Centre de génomique fonctionnelle de l'Université de Sherbrooke, Département de microbiologie et d'infectiologie, Faculté de médecine et sciences de la santé, Université de Sherbrooke
Members of the RNase III family are found in all species studied so far with the exception of archaebacteria where its functions are carried out by the bulge-helix-bulge nuclease. The family is divided into four classes relatives to their functional domains. In bacteria and in baker’s yeast (Rnt1p), only one member of the RNase III is found. In fission yeast and superior eukaryotes, we normally find two RNases III. One is localized in the nucleus (drosha) while the other is in the cytoplasm (dicer). Each RNase III performs distinct and specific functions. Within our work, we used some characteristics to induce specific cleavage in RNA. Our approach allows the control of gene expression at the RNA level in the cell. Moreover, it is possible to apply our approach in vitro using recombinant enzyme. This new technology and its different applications will be discussed in more details.
63 . Julie Parenteau, Mathieu Durand, Bojana Cecez, Valérie Guérin, Andrée-Anne Lacombe, Steeve Véronneau, Raymund J. Wellinger, Sherif Abou Elela and Benoit Chabot
Construction of an intronless Saccharomyces cerevisiae yeast strain
Centre de Génomique Fonctionnelle de l’Université de Sherbrooke, Centre de développement des biotechnologies (CDB) de Sherbrooke, Sherbrooke, Québec, J1E 4K8 Canada
Assessing the role of introns in complex metazoan genomes is a formidable task. Therefore, we have initiated a systematic quest for the function of introns in a less complex organism, the yeast Saccharomyces cerevisiae. This yeast contains ~300 introns in 288 genes. We have classified these genes containing intron in regard to their functions, systematically removed the intron of the genes to create a collection of intronless yeast strains. Until now, only few introns appear to affect cell growth while the vast majority are dispensable. We have also removed the intron from an entire metabolic pathway in a single strain and assayed the effect on growth. Preliminary results indicate that the normal expression and function of the genes coding for yeast cytoskeleton do not require introns.
64 . Ghada Ghazal,Jules Gagnon and Sherif Abou Elela
Identification and analysis of a new class of gene clusters in yeast
Département de Microbiologie et Infectiologie,Faculté de Médecine, Université de Sherbrooke.
Polycistronic mRNAs coding for more than one protein are a common form of gene expression in bacteria. These bacterial mRNAs are expressed from operons of clustered genes that are transcribed from single promoter. In most cases, operons contain functionally related genes that are co-regulated. In general, operons are missing from eukaryotic genomes, where most genes appear to be transcribed individually. In silico analysis of yeast genome indicated the presence of a large number of stem-loop structures that are recognized by the dsRNA specific RNase III (Rnt1p) in intergenic regions that are not associated with known genes. Surprisingly, we found that many of these structures are expressed as part of polycistronic transcripts coding for two-genes clusters that are processed by Rnt1p. This new mechanism of mRNA processing and the functional significance of this finding will be discussed.
65 . Stéphanie Larose, Nancy Laterreur, Ghada Ghazal, Jules Gagnon, Raymund J. Wellinger, Sherif Abou Elela.
RNase III-Dependent Regulation of Yeast Telomerase
Département de Microbiologie, Faculté de Médecine et de Science de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada
Telomerase is a ribonucleoprotein complex essential for maintaining the chromosomes ends, called telomeres. In baker’s yeast, in vivo telomerase activity requires at least four proteins (Est2p, Est1p, Est3p, and Cdc13p) and one RNA species (Tlc1). Telomerase function is tightly regulated during the cell cycle however; the mechanisms that regulate the expression of individual components of the complex are poorly understood. Here we report that yeast RNase III (Rnt1p), a dsRNA specific endoribonuclease, regulates the expression of telomerase subunits and is required for maintaining normal telomere length. Deletion or inactivation of RNT1 induced the expression of Est1, Est2, Est3 and Tlc1 RNAs and increased the telomerase activity leading to elongation of telomeres. In silico analysis of the different RNAs coding for the telomerase subunits revealed a canonical Rnt1p cleavage site near the 3’ end of Est1 mRNA. This predicted structure was cleaved by Rnt1p and its disruption abolished cleavage in vitro. Mutation of the Rnt1p cleavage signal in vivo impaired the cell cycle dependent degradation of Est1 mRNA without affecting its steady-state level. These results reveal a new mechanism that influences telomeres length by controlling the expression of the telomerase subunits.
66 . Mathieu Catala, Maxime Tremblay, Éric Samson, Antonio Conconi and Sherif Abou Elela
Deletion of Yeast RNase III Alters the Chromatin Structure of rRNA Genes
Département de Microbiologie et d’Infectiologie, Faculté de Médecine, Université de Sherbrooke, Québec, Canada
In yeast, the dsRNA specific RNase III (Rnt1p) is required for the processing of pre-rRNA and was shown to co-precipitate with transcriptionally active rDNA repeats. We have found that this enzyme physically interacts with the rRNA polymerase I (Pol I) and that its deletion decreases transcription of rDNA and increases the number of rRNA genes having an open chromatin structure. Normally, 50% of the rRNA genes exhibit open chromatin structure in exponentially growing cells. The number of open rRNA genes is reduced by cell starvation. However, conditions that increase the number of open rRNA genes have not been identified thus far. Here we show that failure in rRNA processing increases the number of rRNA genes with open chromatin. Depletion of ribosomal proteins or factors that impairs Pol I termination did not increase the number of open rDNA, suggesting that changes in the form of rDNA chromatin is specific and not a generic response to ribosome depletion or impaired termination. Our results demonstrate that members of the rRNA maturation machinery may influence the structure of the rRNA genes, and reveal a link between rRNA processing and the conformation of the rDNA chromatin.
67 . Jean-François Lemay1, J. Carlos Penedo2, David M.J. Lilley2 and Daniel A1.
Folding of the Adenine Riboswitch
1- Département de Biologie, Faculté des sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada
2- MSI/WTB Complex, University of Dundee, Dundee, UNited kingdom
The pbuE adenine riboswitch undergoes metal ion-dependent folding that involves looploop interaction. Binding of 2-aminopurine to the aptamer domain strongly correlates with the ability of the loops to interact, and single-molecule FRET studies reveal that folding proceeds via a discrete intermediate. Folding occurs in the absence of adenine ligand, but ligand binding stabilizes the folded structure by increasing the folding rate and decreasing the unfolding rate, and lowers the magnesium ion concentration required to promote the loop-loop interaction. Individual aptamer molecules exhibit great heterogeneity in folding and unfolding rates, but this is reduced in the presence of adenine. In the full riboswitch the adenine binding domain fails to fold because of conformational competition by the terminator stem. Thus riboswitch function should depend on the relative rates of ligand binding and the transcriptional process.
68 . Johans Fakhoury1,2, Delphine Tamara Marie-Egyptienne 1,3 & Chantal Autexier1,2,3
Delineating Species-Specific Telomerase Reverse Transcriptase (TERT) Determinants of Telomerase Function
1- Bloomfield Center for Research in Aging, Lady Davis Institute, Sir Mortimer B. Davis Jewish General Hospital
2- Division of Experimental Medicine, McGill University, Canada
3- Department of Anatomy and Cell Biology, McGill University, Canada
Telomeres cap chromosomal ends, and with telomere-binding proteins, protect chromosomes from being recognized as double strand breaks and processed as such. Telomerase maintains telomere length by adding telomeric repeats. Telomerase is minimally composed of a reverse transcriptase (RT) catalytic subunit (humanTERT & mouseTERT ) and an integral RNA moiety (humanTR & mouseTR) that provides the template (T2AG3) for synthesis. Previous data has shown that marked differences exist in enzymatic activity and processivity between human and mice telomerases. Although telomerase activity can be reconstituted in vitro using combinations of mTERT or hTERT and hTR or mTR, we have shown that mTR-containing complexes exhibit diminished activities in comparison to hTR-reconstituted complexes. Furthermore, mTERT-reconstituted complexes, as well as the hTERT-mTR complex were defective in processivity in comparison to the hTERT-hTR complex. We hypothesize that some of the distinctive features of mouse and human telomerase and telomere biology may result from species-specific differences in TERT-TR interactions, associated proteins, and TERTs that are required for activity of both telomerases. However, immunoprecipitation of TERT components and analysis of coimmunoprecipitated TR revealed similar affinities between TRs and different TERTs. Moreover, activities of telomerase complexes reconstituted in a cellular environment paralleled the activities of the telomerase enzymes in vitro. From the above data, we predict that telomerase activity and processivity is regulated by species-specific conserved TERT sequences and domains. By exchanging domains, species-specific elements that control telomerase activity, processivity, localization, and telomere function will be mapped using both in vitro assays and human and mouse cell lines.
69 . Luc Furic 1, Yoon Ki Kim 2, Lynne E. Maquat 2 et Luc DesGroseillers 1
Identification of the Staufen1-binding Motif in mRNAs
1- Département de Biochimie, Université de Montréal, C.P. 6128 Succursale Centre-Ville, Montréal, H3C 3J7, Canada
2- Department of Biochemistry and Biophysics, School of Medicine and Dentistry, 601 Elmwood Avenue, Box 712, University of Rochester, Rochester, New York, 14642
Staufen is a double-stranded RNA-binding protein involved in mRNA localization and translational control. In Drosophila, Staufen is implicated in the formation of the antero-posterior axis during oogenesis by localizing bicoid and oskar transcripts. In mammals, Staufen1 is a component of RNA granules that move along the microtubule tracts in neurons and is associated with ribosomes and the endoplasmic reticulum in several cell types. To define the role of Staufen, we identified Staufen1-bound mRNAs. To this aim, Staufen1 was immunopurified from mammalian HEK293 cells and isolated mRNAs were used to hybridize microarrays. More than 750 transcripts were enriched at least 5 fold over the negative control and binding of one of them, the ADP-ribosylation factor 1 (Arf1) mRNA, to Staufen1 was confirmed by RT-PCR. To define the Staufen1-binding site (SBS), serial deletions were made from the 3’ end of Arf1 mRNA. The minimal SBS is approximately 200 nucleotides long and is located 60 nucleotides downstream the Arf1 termination codon. Binding of Staufen1 along with the nonsense-mediated mRNA decay factor Upf1 to the SBS induces mRNA decay by a new post-transcriptional mRNA stability mechanism that we termed Staufen1-mediated mRNA decay (SMD). Defining the RNA structural element that permits Staufen1 association with the SBS will allow us to search for a similar motif in other Staufen1-associated mRNAs. It will also help to uncover potential targets of Staufen1-mediated mRNA transport and/or decay and to understand the mechanism of RNA binding specificity by dsRNA binding proteins in mammalian cells.
70 . C. Olivier*1, F. Gallardo*1, R.J. Wellinger2 and P. Chartrand1
Following the intra-nuclear shuttling of the yeast telomerase RNA in situ.
*- These authors have contributed equally to this study.
1- Département de Biochimie, Université de Montréal, Montréal, Québec, H3C 3J7
2- Département de Microbiologie, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4
The length of the telomeres is maintained in eukaryotic cells by a ribonucleoprotein complex called the telomerase. The telomerase complex, in yeast, is composed of a RNA molecule, called TLC1, a reverse transcriptase unit, called Est2p, and two other Est proteins (Est1p and Est3p). In yeast, there are 32 telomeres that assemble into eight to ten foci at the nuclear periphery. Using an in situ hybridization technique to detect the endogenous TLC1 RNA, we have studied the localization of the telomerase complex in a normal yeast cell context. Our results show that the TLC1 RNA can be detected as foci that co-localize with the telomere-associated protein Rap1p. This telomere-associated localization occurs in G1 and S phase and telophase, while in G2 and throughout most of mitosis, TLC1 RNA is segregated in the nucleolus. In order to identify the factors involved in the intra-nuclear shuttling oft the TLC1 RNA, we are currently investigating the impact of the deletion of various nucleolar proteins (Nsr1p, Hmt1p, Gar1p, Pinx1p) and nucleoplasmic proteins (Pif1p, Nab2p) on the TLC1 RNA localization. We are also studying the TLC1 RNA domains that could be implicated in its recruitment and its sequestration in the nucleolus. These results have permitted us to propose a model of intra-nuclear shuttling of the TLC1 RNA, modulated by the cell cycle progression, which could be involved in the regulation of the telomerase activity.
71 . Veronique Trepanier and Luc DesGroseillers
Characterization of the interaction between Staufen1 and mRNA
Département de Biochimie, Université de Montréal
Staufen1 is a double stranded RNA binding protein implicated in mRNA localization and is a component of RNA granules. It is involved in the translational control of mRNAs and is associated with the endoplasmic reticulum and ribosomes. Staufen1 is involved in the Staufen1-mediated mRNA decay (SMD), a novel mechanism that is responsible for the degradation of mRNAs containing a Staufen1 binding site in their 3’UTR. To better understand the role of Staufen1 in mammals, mRNAs that are bound by Staufen1 were identified using microarrays hybridization. Our goal is to characterize the RNA secondary structure that is recognized by Staufen1. Also, we want to compare structures found in different mRNAs to establish a consensus structure. This could allow us to define the specificity of the interaction between Staufen1 and mRNA. One of the Staufen1-bound mRNAs that was identified by microarrays is the mRNA coding for Sec61á1, one of the subunits of the endoplasmic reticulum translocon. First, we confirmed that this mRNA was bound by Staufen1 using immunoprecipitation and RT-PCR. To determine which region of Sec61á1 mRNA is important for the binding, we used deletion mutants of Sec61á and we assessed the capacity of these mutants to bind Staufen1. Our results show that Staufen1 can bind Sec61á mRNA in its 3’UTR. In the 3’UTR, one possible region of 500 nucleotides seems to be important for this interaction. Other deletions and mutations will be necessary to identify more precisely the region implicated in the binding of Staufen1 to this mRNA.
72 . Luc DesGroseillers, Marjolaine Maher Laporte
Characterization of neuronal dendrites Staufen2 ribonucleoprotein (RNP) complexes
Département de Biochimie, Université de Montréal
It has been reported in the past two decades that learning and synaptic plasticity are phenomenons depending on the local translation of specific mRNAs in the dendrites of neurons. The protein Staufen2 is an excellent candidate for studying RNA localization in those events since it is binding double stranded RNA and it is incorporated in ribonucleoprotein particles that move along microtubule in dendrites. Staufen has also been involved in the localization of transcripts during the event of oogenesis and neurogenesis of the drosophila. To better understand de role and the regulation of Staufen2 in these process we need to identify the partners with whom Staufen2 is collaborating. It is also of interest to identify the mRNAs localized by Staufen2. In order to find these interacting partners a proteomic approach has been performed. Also to identify the mRNA found in the Staufen2 complex we used the microarray analysis technique. We confirmed the proteomic partners by co-immunoprecipitation and co-immunofluorescence and some of the microarrays results by RT-PCR.
73 . Rebeca Martinez-Contreras and Benoit Chabot
The positive role of hnRNP F/H in intron definition.
Département de Microbiologie. Faculté de Médecine et des sciences de la santé. Université de Sherbrooke.
hnRNP proteins modulate the alternative splicing of many pre-mRNAs. Previous work on the alternative splicing of the hnRNP A1 pre-mRNA led to the looping out model. In this model, hnRNP A/B proteins bound to intronic sites self-interact to promote exon skipping, repress internal splice sites and facilitate intron definition. Consistent with this model, we have shown that intronic binding sites for hnRNP A/B (ABS) and hnRNP F/H (FBS) stimulate splicing of introns in vivo and in vitro. To examine the generality of the importance of ABS and FBS, we performed a computational analysis for the presence of intronic ABS and FBS at the ends of 156,525 human introns. We found a significant enrichment of putative intronic ABS and FBS near splice sites, supporting the notion that hnRNP A/B and F/H may play a generic role in intron definition. To further explore the contribution of ABS and FBS to intron definition, we selected 30 naturally-occurring introns with putative FBS near both splice sites. All introns were flanking an alternative exon to facilitate monitoring the intron definition event. Total RNA was extracted from cells treated or not with siRNA against hnRNP F/H. Splicing was analyzed by RT-PCR. Out of the 30 events, six were sensitive to hnRNP F/H depletion. We selected the scaffold protein MP1 and the transcription factor NFƒÛƒÒ for further characterization. We have constructed minigenes containing the alternative splicing units and are currently performing mutational and deletional analyses of individual and combined FBS to understand their contribution to intron definition
74 . Lulzim Shkreta1, Ulrike Froehlich2, Eric Paquet2, Johanne Toutant1, Benoit Chabot1,2
Alternative splicing of human apoptotic genes is a major target of chemotherapeutic drugs
1- Département de microbiologie et d’infectiologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke
2- Centre de génomique fonctionnelle de Sherbrooke, Québec, Canada
Apoptosis is the essential control pathway of cell proliferation that is often disrupted in cancer cells. For this reason the apoptotic pathways have emerged as promising targets for cancer treatments and their activation represent the core action of mainstream chemotherapeutic drugs. The majority of the genes, encoding the more than 200 pro- and anti-apoptotic proteins involved in apoptotic control are alternatively spliced. Regulatory proteins of opposing functions are often produced from a common pre-mRNA. Thus, alternative splicing of apoptotic genes is of a crucial importance for apoptotic regulation. We have undertaken a large-scale systematic analysis of the effects of chemotherapeutic drugs on the alternative splicing of apoptotic genes. Twenty of the most common chemotherapeutic drugs were used to treat one transformed (293 cells) and several cancer cell lines (PA-1, SKOV-3, MCF-7 and PC-3), representing ovarian, mammary gland and prostate tumors. The splicing profile of 96 alternative splicing units from the apoptotic genes were obtained by RT-PCR following treatment of cells with various drugs. Bioinformatics processing of the data revealed common targets for different drugs as well as targets shared by different cell lines. Many drugs induced a splicing shift toward pro-apoptotic isoforms. Generally, 293 cells were more sensitive than the cancer cells lines MCF-7, PC-3, PA-1 and SKOV-3 although the majority of the drugs affected the alternative splicing of bcl-x. Overall, the changes in the alternative splicing profile of apoptotic genes induced by drugs were largely cell-line specific.
75 . Karine Boulay and Luc DesGroseillers
Characterization of the association between Stau1 and ribosomes
Département de Biochimie, Université de Montréal, Montréal, QC, Canada
mRNA localization represent a mechanism to target cytoplasmic proteins to specific cellular regions. The efficiency of this mechanism relies on a tight regulation of translation during the process. We are interested in Staufen, a double-stranded RNA-binding protein that is involved in RNA localization either in invertebrates or mammals. Some evidences indicate that Staufen could play a role in regulation of translation since it was shown to be associated with ribosomes. Staufen determinants implicated in this interaction were mapped. Moreover, we have recently shown that Staufen can facilitate translation of repressed mRNAs. We wanted to characterize the association between hStau1 and ribosome in order to understand how it could participate in translation regulation of targeted transcripts. We performed cytoplasmic fractionation assays of 293T cells with S100-P100 technique and sedimentation on sucrose gradients. The first technique allowed us to determine that hStau1 interacts with ribosome through ionic interactions independently of membranous structures. We were also able to evaluate the affinity of Staufen determinants for the ribosome with mutants of the protein. Sedimentation on sucrose gradients experiments showed that hStau1 interacts mainly with large ribosomal subunit independently of its RNA-binding capacity. In addition, hStau1 is associated to actively translating polyribosomes. We are now attempting to identify protein partners that mediate the association with ribosome by using crosslinking agents prior to immunoaffinity purification assays. We believe that investigations on these proteins will elucidate the mechanisms by which Staufen could contribute to the efficiency of mRNA localization and translation regulation.
76 . Jean-François Jacques1, Soojin Jang2, Karine Prévost1, Maxime Desmarais1, James Imlay2, Eric Massé1
RyhB small RNA modulates the free intracellular iron pool and is essential for normal growth during iron limitation in Escherichia coli
1- Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Groupe ARN. Université de Sherbrooke, Sherbrooke, Québec, Canada
2- Department of Microbiology, University of Illinois, Urbana, Illinois 61801
The small RNA RyhB has recently been shown to negatively regulate a number of mRNAs encoding dispensable iron-containing proteins in Escherichia coli. The resulting decrease in the synthesis of iron-containing proteins is expected to spare iron and ensure its availability for Fe-requiring proteins that are indispensable. Indeed, the expression of RyhB from a heterologous promoter activates the iron-sensing repressor Fur, which suggests an increase in the pool of free intracellular iron (iron-sparing model). In accordance with these observations, we report here that RyhB expression increases the concentration of free intracellular iron, as shown by direct measurements of the metal in whole cells by electron paramagnetic resonance spectroscopy (EPR). Our data also suggest that iron-sparing originates from rapid uptake of extracellular iron and not from already internalized metal. Furthermore, RyhB is shown to be essential for normal bacterial growth and survival during iron starvation, which is consistent with previous data describing the function of the small RNA. Overall, our data demonstrate that, by regulating synthesis of non-essential Iron-containing proteins, the small RNA RyhB acts to ensure that limiting free intracellular iron is used for critical proteins.
77 . Kaitlin Soye1, Sébastien Lainé1, Patricia Landry2, Jean-Pierre Perreault2 and Anne Gatignol1
Decreased replication of HIV-1 by targeting the viral proteins Tat and Rev, and host cell TAR-RNA Binding Protein (TRBP) with delta ribozymes
1- Lady Davis Institute for Medical Research, McGill University, Montréal, Québec
2- Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec
HIV-1 replication can be depressed using RNA-based methods such as antisense RNA, RNA decoys, RNA interference and ribozymes (Rz). Among the different Rz, the delta Rz (dRz) from Hepatitis D virus has not been tested against HIV-1. TRBP exists in two forms, TRBP 1 and TRBP 2, and binds to the TAR (trans-activation-response) RNA region in the HIV-1 LTR (long terminal repeat) sequence. HIV-1 replication is decreased in astrocytic cell lines compared to lymphocyte cell lines due to a weak TRBP expression suggesting that a decrease of TRBP could be a new approach to decrease viral replication. New dRz have been designed. The SOFA (specific ON/OFF adapter) dRz has a blocker, biosensor and stabilizer region making it more reliable and specific. dRz were designed to target HIV-1 Tat (dRzTat) and Rev (dRzRev), as well as TRBP 1 (dRzTRBP1) and TRBP 2 (dRzTRBP2). Using luciferase assays, dRzTat decreases the basal level of LTR expression by 50%, and when various dRzTat are used in combination, the expression of the LTR is decreased by 60%. When used to target HIV-1 the dRzTat decreases viral replication by 60%. Additionally, the dRzRev decreases viral replication by 60%. The dRzTRBP1 and 2 show a 50% decrease of basal LTR activity. When the dRz’s against TRBP are used in combination the effectiveness is not increased. In the presence of HIV-1, the dRzTRBP1 decreases viral replication by 50%. The dRz have the ability to be a potent tool to reduce HIV replication.
78 . Graeme AM Nimmo1,2, Ryan J Ward1,2, Maria A Cerone1,3, and Chantal Autexier1,2, 3
The role of the human telomerase RNA in tumorigenesis
1- Bloomfield Center for Research in Aging, Lady Davis Institute, Sir Mortimer B. Davis Jewish General Hospital
2- Division of Experimental Medicine, McGill University, Canada
3- Department of Anatomy and Cell Biology, McGill University, Canada
Telomerase, the ribonucleoprotein that maintains the ends of linear eukaryotic chromosomes is comprised of the protein subunit telomerase reverse transcriptase (hTERT, in humans) and the telomerase RNA (hTR). Traditionally, reactivation of telomerase was thought to be required mainly to impart an indefinite lifespan. Recently, however, several studies have suggested that telomerase may mediate telomere lengthening-independent functions. Some evidence indicates that certain of these functions may not require the telomerase RNA or a catalytically active enzyme. We are interested in characterizing how telomerase may contribute to tumorigenesis independently of it role in telomere addition. Here we have assessed the requirement of hTR for this ‘non-classical’ function of telomerase. We have taken advantage of well defined cell lines that do not express telomerase but are able to maintain their telomeres via another mechanism: ALT (alternative lengthening of telomeres). It has been previously established that ALT cells expressing hTR require both oncogenic Ras (RasV12) and hTERT to form tumours in nude mice. We have used the ALT cell line VA13 (hTERT-; hTR-) to generate clones that stably express hTERT and RasV12 in the presence or absence of hTR, and will determine the ability of these clones to form tumors using an in vitro hard-agarose assay as well as an in vivo mouse xenograft model. We expect that clones that do not express hTR will form tumors if hTR is not required for this ‘non-classical’ function of telomerase.
79 . F.Gallardo1*, C.Olivier1*, R.J. Wellinger2 and P.Chartrand1
Following the nucleo-cytoplasmic shuttling of the yeast telomerase RNA.
*- These authors contribute equally to this work.
1- Département de Biochimie, Université de Montréal, Québec, H3C 3J7.
2- Département de Microbiologie, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4.
In yeast, the telomerase holoenzyme, responsible for telomere elongation, is a ribonucleoprotein complex composed of three proteins named Estp and an RNA component named TLC1. Its recruitment to telomeres has been shown to be mediated by the Ku70/Ku80p complex, but little is known about its intracellular trafficking in vivo. By using FISH experiments against the endogenous form of the TLC1 RNA, we have shown that the deletion of EST2 or KU70, as well as the impairment of the binding of Ku70p on TLC1, leads to the accumulation of the TLC1 RNA in the cytoplasm. These results suggest that the TLC1 RNA goes through a cytoplasmic step in its maturation, and comes back to the nucleus before its recruitment to telomeres. We have also deleted the methyltransferase TGS1, known to be involved in the CAP modification of snRNA and possibly TLC1, and found that the telomerase RNA accumulate inside the nucleolus. In order to determine which pathways are involved in the nucleo-cytoplasmic shuttling of the TLC1 RNA, we are currently testing a set of mutants of the major RNA export pathways in S.cerevisiæ: Mex67p/Dbp5p for polyA-mRNA export, Crm1p for NES-containing protein and Nsp1p/Rps15p for ribosomal RNA. This study will allow the identification of factors required for the dynamics of the telomerase complex in vivo.
80 . Casey C. Fowler, Eric D. Brown, Yingfu Li
Flow cytometry based selection for artificial riboswitches
McMaster University
Riboswitches are RNA regulatory elements that directly bind a particular ligand and respond by altering the expression of downstream genes. Riboswitches are desirable instruments for those seeking to engineer inducible gene expression systems due to their relatively compact and straightforward nature. In recent years a few groups have begun to exploit this potential; however a reliable means to create artificial riboswitches that respond to a target of choice is still lacking. Here we present our progress toward developing such a scheme for use in prokaryotic organisms. We have used an established RNA aptamer connected to a transcriptional terminator by a partially random linker in order to create a library of candidate riboswitches. We then cloned these sequences into a plasmid within the 5' UTR of GFP and transformed this library into E. coli cells. Using fluorescence assisted cell sorting (FACS) both in the presence and in the absence of the aptamer's cognate ligand, we isolated sequences from the library that act as riboswitches. Using lower throughput GFP assays we further refined the search and have isolated several riboswitches with significant, ligand-responsive enhancements in reporter activity. We hope to build on this initial success by creating riboswitches that could be used in gene regulatory networks or that act as indirect reporters for cellular enzymatic inhibition assays.
81 . Simon A McManus, Yingfu Li
Characterization of a Kinase Deoxyribozyme with an Entwined Helix-Quartet Structure
McMaster University
Here we report a deoxyribozyme with a unique structure that contains a two-tiered guanine quadruplex interlinked to a Watson-Crick duplex. Through in vitro selection, sequence mutation, and methylation interference, we show the presence of both the two-tiered guanine quartet and two helical regions contained in the active structure of this deoxyribozyme. Interestingly, one GG element of the quartet is part of a hairpin loop within one of the identified helical regions. Circular dichroism analysis showed that antiparallel quartet formation was dependent on this helix. To our knowledge, this is the first report of a pseudoknot nucleic acid structure that involves a guanine quartet. Our finding indicate that guanine quartets can be a part of complex structural arrangements, increasing the likelihood of finding more complex guanine quartet arrangements in biological systems.
82 . Jeff Lam, Johanna Withers, Yingfu Li
Characterization of a deoxyribozyme capable of cleaving a pyrimidine-pyrimidine junction reveals a complex secondary structure
McMaster University
Several small RNA-cleaving deoxyribozyme motifs have been reported for efficient cleavage of purine-purine or purine-containing dinucleotide junctions. Most of these deoxyribozymes are incapable of cleaving any pyrimidine-pyrimidine junctions. We hypothesize that such an activity may require larger deoxyribozymes with more complex secondary (and tertiary) structures, which are often out competed by small RNA-cleaving motifs during standard in vitro selection experiments. To test this hypothesis, we carried out a modified in vitro selection study to derive new classes of deoxyribozymes by only employing a CrT junction (a ribo-C embedded in a DNA chain) as a representative pyrimidine-pyrimidine junction. Our results show a novel deoxyribozyme effectively cleaves a pyrimidine-pyrimidine junction at a k(obs) of ~0.35 min^-1. Secondary structure study of the DNAzyme revealed a four-way junction motif enclosing an additional helix embedded in its catalytic core. Given that many natural ribozymes performing difficult chemical reactions often require complicated structural arrangements, our observation provides an example that catalytic DNA can also form complex structures to catalyze comparable reactions.
83 . William Chiuman, Yingfu Li
In vitro evolution of new deoxyribozymes by sampling the sequence variants of a catalytic DNA
McMaster University
Survival of the fittest is the basis of Darwinian evolution. Here we recount a story of how a deoxyribozyme, consisted of the most common and the simplest three-way junction framework, evolved into better catalysts that have a similar five-way junction motif. The transition was shown to be a switch rather than a gradual progression. The new motif, surprisingly, only took five selection cycles to be detected and another four to dominate the evolving population. Beside just a story, we have also illustrated the idea of deriving a rare structural motif by sampling the sequence variants of a functional nucleic acid.
84 . Marie-Éve Stébenne and Brendan Bell
Mechanisms of pro-apoptotic alternative splicing of the taf6 gene
Université de Sherbrooke
TFIID is an RNA polymerase II general transcription factor that plays a key role in the regulation of the expression of protein-coding genes. TFIID is the central core promoter recognition factor and is composed of TATA-binding protein (TBP) and 14 other TBP-associated factors (TAFs). Recent work from our laboratory has suggested that TFIID function can be changed in certain types of apoptosis (programmed cell death). TAF6delta is an apoptosis-specific TFIID subunit produced by alternative splicing of the TAF6 pre-mRNA. Apoptosis is an active form of cellular death that is deregulated in several human disease states, notably cancer. Using modified antisense RNA oligonucleotides we have shown that forcing splice site choice of of TAFdelta leads to apoptosis in several cancer cell lines. Given that TAFdelta can control life versus death decisions of cancer cells, we have begun to dissect the cis-acting RNA elements of the taf6 pre-mRNArequired for expression TAF6delta by alternative splicing. We have developed a taf6 minigene construction that faithfully recapitulates the splicing pattern of the endogenous taf6 gene in transfected HeLa cells. Using site-directed mutagenesis, we have identified two regulatory elements in the taf6 pre-mRNA. One element is composed of two well-conserved CCC motifs in intron 2, the mutation of which results in diminished TAF6delta production. A second element is found in alternative exon 2 appears to be critical for the selection of TAF6alpha alternative splicing. Our ongoing experiments to define the mechanisms of TAF6 alternative splicing will be presented.
85 . Emmanuelle Wilhelm and Brendan Bell
Modified RNA antisense oligonucleotides reveal a role for the delta splice variant of TAF6 in gene expression and apoptosis.
Université de Sherbrooke
Apoptosis is an active form of cell death required for normal development and tissue homeostasis. Deregulated apoptosis is implicated in many disease states including cancer, neurodegenerative diseases and AIDS. Changes in gene expression can play a key role in the induction of apoptosis, yet the molecular mechanisms that control gene expression in response to cell-death signals remain poorly understood. Our previous work has suggested a model whereby apoptotic signals can be coupled to changes in gene expression programs through the function of the general RNA polymerase II transcription factor TFIID. TFIID is composed of TATA-binding protein plus 14 TAFs (TBP-associated factors). TAF6delta is a functionally unique isoform of the TFIID subunit TAF6, that is induced in certain forms of apoptosis. We have employed transfection of 2’-O-Methyl phosphorothioate antisense RNA oligonucleotides to specifically induce expression of the TAF6delta mRNA. Induction of endogenous TAF6delta by this technique results in apoptosis of cells, independently of the expression of the p53 tumor suppressor gene. Microarray experiments further revealed that forced TAF6delta expression results in cellular gene expression programs including the increased expression of multiple well-known pro-apoptotic genes. Our results show that TAF6delta can orchestrate gene expression programs to control life or death decisions of human cells.
86 . Isaac M. Nzaramba, Emmanuelle Wilhelm, Brendan Bell
HIV Core Promoter: New CTGC Motifs Responsible of Its Specificity.
Université de Sherbrooke
A major obstacle to the eradication of AIDS is the capacity of latent virus to evade immune surveillance and available anti-HIV drugs. Latency is a normal step in the HIV life cycle which occurs when gene expression is repressed at the post-integration level. Transcription of the HIV genome is strictly controlled by host cell transcription factors together with the virally encoded Tat protein. The HIV Tat protein specifically trans-activates the initiation and the elongation steps of trascription thus, increasing the rate of HIV gene expression. In addition to the nascent RNA TAR stem-loop structure, the HIV core promoter is specifically required for full Tat trans-activation of the HIV promoter. To define the cis-acting DNA sequences specifically required for Tat-responsive transcription, we have extensively mutated the HIV-1 core promoter and tested these mutations in transfection assays. We have identified 3 new 5’-CTGC-3’ DNA motifs that flank the HIV “TATA-box” that are necessary for the Tat trans-activation of the core promoter in HeLa cells. In addition, the observation of cellular pre-initiation complex proteins assembled on the HIV core promoter by the EMSA technique revealed alterations in the formation of pre-initiation complexes containing TBP and a subset of TAFs when the CTGC motifs are mutated. Taken together, these findings demonstrate a novel role for the CTGC DNA motifs of the HIV core promoter in the formation of functional distinct pre-initiation complexes that respond specifically to Tat trans-activation and shed new light on HIV gene expression.
87 . Patricia Bouchard, Geneviève Desjardins, Dean Campbell et Pascale Legault
NMR and thermodynamic studies of metal binding by stem-loop V from the Neurospora VS ribozyme
Université de Montréal, University of Georgia
We have studied the NMR structure of stem-loop V (SL5) from the Neurospora VS ribozyme. Stem-loop V is the acceptor RNA for stem-loop I; the tertiary interaction involving these two stem-loops is important for substrate recognition and activation in the VS ribozyme. High-resolution NMR structures of SL5 have been obtained both in the absence and in the presence of Mg2+. Our studies demonstrate that Mg2+ induce a conformational change in which the U-turn motif of SL5 adopts more characteristics of canonical U-turn structures. In addition, the magnesium-bound SL5 loop structure present a novel recognition surface for stem-loop I binding. Using paramagnetic NMR methods, we have localized four magnesium ions associated with the SL5 loop. We have also analyzed the contribution of metal ions (Na+ and Mg2+) to the loop stability of SL5. Thermodynamic parameters were determined from UV melting curves for the SL5 RNA fragment used in our NMR structural investigations and for a mutant RNA hairpin with no U-turn loop structure. The dependence of the structural stability of these RNA hairpins on salt concentration was defined via the slope of Tm/log[ion] plots. Data obtained for the wild-type and mutant RNAs allow us to estimate the contribution of metal ions to the structural stabilities of the U-turn loop in SL5. Our results help further understand the effect of Mg2+ on the stem-loop V U-turn structure and on the formation of the stem-loop I / stem-loop V interaction.
88 . M. Asif Ullah and Jean-Pierre Perreault
Inhibition of Hepatitis B Virus Replication by SOFA Delta-Ribozyme
Département de biochimie, Université de Sherbrooke
Human hepatitis B virus (HBV) infects chronically over 400 million peoples globally, and responsible for more than 1 million deaths annually. RNA cleavage of virus specific genes has emerged as a potential antiviral mechanism. SOFA (specific on/off adapter) delta ribozyme activity is being successfully tested in vitro. The ultimate goal was to verify the HBV inhibition profile in cultured mammalian cells by introducing SOFA-delta ribozymes. Here we present a significant reduction in replicated HBV DNA and RNA levels with plasmids expressing SOFA delta ribozymes and HBV genome in cultured cells co-transfected. Sustained level of HBV DNA and RNA inhibition were analyzed upto 10 days post transfection by Immuno-Capture PCR and Northern. Secreted HBV surface antigen (HBsAg) and core antigen (HBcAg) in cultured cells measured by ELISA were also found reduced. Our results represents the enhanced potential of the target dependent module of ribozymes with improved activity and specificity in cellular environment.
89 . Laurent Chatel-Chaix1,2, Levon Abrahamyan2, Céline Fréchina1, Andrew J. Mouland2,3,4 and Luc DesGroseillers1
The host RNA-binding protein Staufen1 participates in HIV-1 assembly in live cells by influencing pr55Gag multimerization
1- The Département de biochimie, Université de Montréal;
2- HIV-1 RNA Trafficking Laboratory, Lady Davis Institute for Medical Research-Sir Mortimer B. Davis Jewish General Hospital;
3- Department of Microbiology & Immunology;
4- Department of Medicine, McGill University, Montréal, Québec, Canada.
Human immunodeficiency virus type 1 (HIV-1) requires the sequential activities of virus-encoded proteins during replication. As an obligate parasite, the activities of several host cell proteins and machineries are critical to the completion of virus assembly and the release of fully infectious virus particles from cells. One of these proteins, the double-stranded RNA-binding protein Staufen1 (Stau1), selectively associates with the HIV-1 genomic RNA and the viral precursor Gag protein, pr55Gag. In this report, we tested whether Stau1 modulates pr55Gag assembly using a new pr55Gag oligomerization assay based on bioluminescence resonance energy transfer (BRET) in both live cells and extracts after cell fractionation. Our results show that both over-expression and knockdown of Stau1 increase the pr55Gag-pr55Gag BRET levels, suggesting a role for Stau1 in enhancing pr55Gag oligomerization during assembly. Although we show that Stau1 and pr55Gag interact in both cytosolic and membrane compartments, this effect of Stau1 on pr55Gag oligomerization was only observed in membranes, a cellular compartment in which pr55Gag assembly primarily occurs. Consistently, membrane-associated detergent-resistant pr55Gag complexes were formed when Stau1 expression levels were modulated. Furthermore, expression of Stau1 harbouring a vSrc myristylation signal lead to a 6.5-fold enrichment in membranes and a corresponding enhancement in the Stau1-mediated effect on pr55Gag-pr55Gag BRET demonstrating that Stau1 acts on assembly when targeted to membranes. Our results indicate that Stau1 influences HIV-1 assembly by modulating pr55Gag-pr55Gag interactions, a key event that drives virus assembly, as shown in a live cell interaction assay. This likely occurs when Stau1 interacts with membrane-associated assembly intermediates.