snoRNAs are a large family of small non-coding RNAs which guide the post-transcriptional modification of specific positions in target RNAs (mainly in rRNA but also in snRNAs and tRNAs). Two main classes of snoRNAs have been described, the box C/D snoRNA which catalyze 2' O-methylation of their targets, and the box H/ACA snoRNAs which guide the pseudouridylation of their targets (1-3).
Box C/D snoRNAs
Box C/D snoRNAs are characterized by the presence of conserved boxes C (sequence: RUGAUGA) and D (sequence: CUGA) near the extremities of the molecule. Additional boxes C' and D' with the same consensus motif as boxes C and D respectively but often less well-conserved are also observed. The guide region, with complementarity to the target is found immediately upstream of the box D and/or the box D' (1-3). Boxes C and D are believed to come into close proximity in the folded molecule (Fig. 1), forming a structural motif called the kink-turn or k-turn, which is characterized by the non-canonical interaction of G-A base pairs and resulting in a sharp kink in the molecule.
By analysis of deep-sequencing of small RNAs, we identified two general groups of box C/D snoRNAs differing in the exact end of their sequence with respect to their boxes C and D. The short box C/D snoRNAs start 4-5 nt upstream from the box C and end 2-3 nt downstream from the box D while the long box C/D snoRNAs start 5-6 nt upstream from the box C and end 4-5 nt downstream from the box D. These two groups of box C/D snoRNAs display differential biogenesis dependencies and binding preferences to core box C/D binding proteins, and a subset depend on the splicing factor RBFOX2 for accumulation, as described in (4). Our data suggest that box C/D snoRNAs can be divided into at least two groups with distinct maturation and functional preferences (Figure 2).
(1) Kiss, T. (2001) Small nucleolar RNA-guided post-transcriptional modification of cellular RNAs. Embo J, 20, 3617-3622.
(2) Matera, A.G., Terns, R.M. and Terns, M.P. (2007) Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs. Nat Rev Mol Cell Biol, 8, 209-220.
(3) Watkins, N.J. and Bohnsack, M.T. (2012) The box C/D and H/ACA snoRNPs: key players in the modification, processing and the dynamic folding of ribosomal RNA. Wiley interdisciplinary reviews. RNA, 3, 397-414.
(4) Deschamps-Francoeur et al (2014) Identification of discrete classes of small nucleolar RNA featuring different ends and RNA binding protein dependency, NAR in press.
Over the past forty years, numerous signals and localization mechanisms have been uncovered including protein targeting motifs as well as protein interaction domains and post-translational modifications regulating localization. Protein localization regulation is also multi-layered and dynamic as witnessed by recent large-scale microscopy and proteomics projects which have identified large numbers of proteins localizing in more than one cellular compartment. The mislocalization of proteins caused by the disruption of localization mechanisms is a molecular hallmark of numerous diseases. We are interested in the study of the combinatorial regulation of proteins, including the importance and contribution of individual mechanisms. We are currently investigating to what extent the presence of targeting motifs in a protein is regulated at a transcriptional and post-transcriptional level, by considering deep-sequencing datasets.
Determining the contribution of alternative splicing to cancer biology
Alternative splicing (AS) is a post-transcriptional process whereby the introns and exons of a single gene transcript are differentially spliced to yield multiple mature mRNAs. In eukaryotes, AS plays a central role in both protein diversity and post-transcriptional gene regulation. AS within mRNA coding regions can lead to multiple, functionally diverse protein isoforms from a single gene transcript. AS can also act to introduce or remove regulatory elements to affect the translation, localization or degradation properties of an mRNA.
The majority of human genes are thought to undergo alternative splicing. The capacity of alternative splicing to change the protein function and cellular phenotype made it a prime suspect for human disease. Indeed, it is now linked to many well-known diseases like cystic fibrosis, thalassemia and spinal muscular atrophy.
We have developed tools for discovering and studying the function of cancer associated splice variants. Using these newly developed bioinformatics / genomics techniques we identified two different groups of splicing events required for cell survival, cell cycle progression and apoptosis.
Currently, the work in the laboratory aims at linking splicing factors with cancer specific splicing program and identifies the specific functions of splice variants in the tumor microenvironment and the cell cycle.
Michelle L, Cloutier A, Toutant J, Shkreta L, Thibault P, Durand M, Garneau D, Gendron D, Lapointe E, Couture S, Le Hir H, Klinck R, Elela SA, Prinos P, Chabot B. Proteins associated with the exon junction complex also control the alternative splicing of apoptotic regulators. Mol Cell Biol. 2012 Mar;32(5):954-67.
Venables JP, Klinck R, Koh C, Gervais-Bird J, Bramard A, Inkel L, Durand M, Couture S, Froehlich U, Lapointe E, Lucier JF, Thibault P, Rancourt C, Tremblay K, Prinos P, Chabot B, Elela SA. Cancer-associated regulation of alternative splicing. Nat Struct Mol Biol. 2009 Jun;16(6):670-6.
Venables JP, Brosseau JP, Gadea G, Klinck R, Prinos P, Beaulieu JF, Lapointe E, Durand M, Thibault P, Tremblay K, Rousset F, Tazi J, Abou Elela S, Chabot B. RBFOX2 is an important regulator of mesenchymal tissue-specific splicing in both normal and cancer tissues. Mol Cell Biol. 2013 Jan;33(2):396-405.
Prinos P, Garneau D, Lucier JF, Gendron D, Couture S, Boivin M, Brosseau JP, Lapointe E, Thibault P, Durand M, Tremblay K, Gervais-Bird J, Nwilati H, Klinck R, Chabot B, Perreault JP, Wellinger RJ, Elela SA. Alternative splicing of SYK regulates mitosis and cell survival. Nat Struct Mol Biol. 2011 Jun;18(6):673-9.
Brosseau JP, Lucier JF, Lapointe E, Durand M, Gendron D, Gervais-Bird J, Tremblay K, Perreault JP, Elela SA. High-throughput quantification of splicing isoforms. RNA. 2010 Feb;16(2):442-9.
G-quadruplexes (G4) are structures found among guanine rich nucleic acids (1). When folded within an RNA, the G4 was shown to have gene regulation capabilities (2-5). The G4 is a tetra-helical structure containing 3 loops linking the 4 helical strands as described by the G>2N1-7G>2N1-7G>2N1-7G>2 motif. This motif was long used to predict G4s within nucleic acids (6) but recent studies has shown the folding of G4s within sequences not matching the motif and sequences tallying the motif not folding into G4s (7).
We are developping tools to ease the work of experimenters in their search for a greater RNA G4 comprehension and the characteristics shared by sequences that folds into the structure.
1. Gellert M., Lipsett M. N. and Davies D. R. (1962) Helix formation by guanylic acid. Proc. Natl. Acad. Sci. United States Am., 48, 2013-2018.
2. Kumari S., Bugaut A., Huppert J. L., et al. (2007) An RNA G-quadruplex in the 5’ UTR of the NRAS proto-oncogene modulates translation. Nat. Chem. Biol., 3, 218–221.
3. Huppert J. L., Bugaut A., Kumari S., et al. (2008) G-quadruplexes: the beginning and end of UTRs. Nucleic acids Res., 36, 6260–6268.
4. Beaudoin J. D. and Perreault J. P. (2010) 5’-UTR G-quadruplex structures acting as translational repressors. Nucleic acids Res., 38, 7022–7036.
5. Beaudoin J. D. and Perreault J. P. (2013) Exploring mRNA 3’-UTR G-quadruplexes: evidence of roles in both alternative polyadenylation and mRNA shortening. Nucleic acids Res., 41, 5898–5911.
6. Huppert J. L. and Balasubramanian S. (2005) Prevalence of quadruplexes in the human genome. Nucleic Acids Res., 33, 2908–2916.
7. Beaudoin J. D., Jodoin R. and Perreault J. P. (2014) New scoring system to identify RNA G-quadruplex folding. Nucleic acids Res., 42, 1209–1223.