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Alternative pre-messenger RNA splicing is responsible for the production of possibly millions of proteins encoded by 20 000 different human genes. The process that dictates the combination of different portions of sequences to fabricate distinct messenger RNAs is under firm control but is poorly understood. To understand how this complexity is generated and regulated, we are studying how the selection of splice site occurs in Bcl-x, a gene whose splicing products decide if a cell lives or dies. We have identified several controlling elements and proteins that dictate differential splice site usage and we are planning to understand the contribution and mechanism of action of each factors when cells are submitted to a variety of signals, drugs and stresses. Insights into this process may help develop novel strategies to monitor and combat human diseases including cancer.
Genetic variation between individuals can lead to simple and complex phenotypic traits including susceptibility to disease and response to treatment. The genetic differences between individuals can range from gross alterations in the karyotype to single nucleotide changes. Many human genes contain arrays of short (30-200 bp) repetitive units arranged in tandem arrays called minisatellites or VNTRs. These VNTRs are highly unstable in the human population and changes can occur within one generation. Many VNTRs are located in genes, and portions of them can be coding. Possibly reflecting their repetitive nature, we have shown that pre-messenger RNAs harboring VNTRs are alternatively spliced with a frequency that is considerably superior to regular genes. Our hypothesis is that mutations occurring in VNTR-containing genes may alter their alternative splicing, offering opportunities for producing and testing new variants that may confer advantages for growth, adaptation and survival in a changing or stressing environment. The other side of this evolutionary coin is that the same process may also confer negative properties like susceptibility to diseases. Our objectives are to determine (i) whether the intrinsic instability of VNTRs has a permanent effect on alternative splicing, and (ii) whether induced genomic instability preferentially acts on VNTRs to affect the alternative splicing of the genes that harbor them.
In spite of its imposing biological relevance and undeniable role in human diseases, tissue-specific annotation of alternative splicing (AS) has not been done systematically and very little is known about the individual functions of splice isoforms. While expression profiling of cancer has provided clues about key genes involved in tumourigenesis, there is clearly a need for new investigations that will map with more precision the topology of this disease, link disease-specific gene expression patterns to oncogenic function, and offer new avenues for diagnosis, treatments and follow-up. Several projects focus not only on the discovery of AS variants to be used as biomarkers for the recurrence of cancer, but also on the identification of functionally relevant forms that can offer therapeutic value. Our research program also provides a systematic approach towards understanding the fundamental principles responsible for alternative splicing dysfunction in cancer.Alternative splicing studies in breast and ovarian cancer. During the last 5 years we made important progress in understanding the role of alternative splicing in the biology of breast and ovarian cancer by uncovering a multitude of cancer-associated variants, some of which required for cancer cell viability in vitro. Our main discoveries have been to uncover a well-defined cancer-dependent regulatory network and a clear link between AS and cell survival. Our work generated a large number of potential cancer markers and opened the way for in depth analysis. We have built a team with considerable expertise in variant analysis, established platforms to monitor alternatively splicing events (ASEs), their regulatory factors, and perform phenotypic assessment. Progress can be summarized as follows:
a) Establishing systems for detecting cancer-associated splice variants:
We have developed a platform for the detection of AS using endpoint PCR, and have used it to identify splicing events in ovarian and breast cancers (Klinck et al., 2008. Cancer Res. 68:657, Venables et al., 2008. Cancer Res. 68:9525). To detect ASEs in clinical samples we developed a new high-throughput real time PCR procedure (Brosseau et al., 2010. RNA 16:442).
b) Role of splicing factors in controlling the expression of cancer-associated splicing events:
We conducted the first large screen of cancer-associated splicing events and identified RBFOX2 as a major regulator of AS in cancer cells (Venables et al., 2009. NSMB 16:670) (Venables et al. 2013 MCB 33:396-405.
c) Functional annotation of splicing isoforms:
We developed a new method to reprogram AS decisions without affecting overall gene expression, and used it to show that changing the splicing pattern of SYK kinase impairs cell-cycle progression and anchorage-independent growth. We have implicated AS as an important modulator of the EGF/MAPK pathway, and have provided a robust demonstration of the role of splice variants in tumourigenesis (Prinos et al., 2011. NSMB 18:673).
The RNomics Platform provide state-of-the-art nucleic acid annotation and quantification services, expertise, and training to researchers at the Université de Sherbrooke and worldwide. It was developed to support internal research programs on alternative splicing (AS) in human cells and tissues, the platform currently provides a vast array of deep sequencing and PCR-based services, and expertise related to analysis of the transcribed genome. The platform has developed an automated computational and experimental system for the high-throughput analysis of AS and gene expression using standard and quantitative PCR (qPCR). The contributions of the platform have included the identification of hundreds of changes in AS associated with breast and ovarian cancers, which has provided novel insight into the biology of cancer. The platform has also been instrumental in identifying AS events linked to various conditions such as stress, development, aging, response to infection, and environmental changes. Furthermore, the platform has been used to identify genes whose AS is regulated by individual RNA binding proteins such as hnRNP and snRNP proteins, RBFOX2 and EJC components, hence revealing networks of AS regulation. Recently, the platform’s technology has been expanded to include next-generation sequencing and analysis tools. Several sequencing projects are currently being carried out, including RNA-Seq analysis of snoRNA and pre-snoRNAs in ovarian cancer cell lines and yeast, RNA-Seq and ChIP-Seq studies in human cell lines.
The platform is equipped with 3 next-generation sequencing instruments (IonTorrent PGM, Roche GS Junior, and Illumina MiSeq), 6 PCR and 6 qPCR thermocyclers, 5 capillary electrophoresis instruments, 2 liquid handling robots and all peripheral equipment required for high throughput DNA and RNA extraction, preparation and analysis from tissue and cell culture sources. The workflow is supported by a laboratory information management system (LIMS) developed specifically for the platform, which includes a web application which allows researchers to consult and download data via a secure Internet connection. Sample data can be consulted here: http://palace.lgfus.ca/pcrreactiongroup/list/85.
The platform occupies 120 m2 in the PRAC building on the health campus of the Université de Sherbrooke. Benoit Chabot, Sherif Abou Elela and Roscoe Klinck developed the platform and are among its principal users. Klinck is the platform manager and adjunct professor in the Department of Microbiology at the Faculté de Médecine de l’Université de Sherbrooke (FMSS).
Key Achievements of the RNomics Platform:
• State-of-the-art next-generation sequencing, bioinformatics and high-throughput automation facilities maintained by 4 highly qualified personnel.
• Over 100 novel putative ovarian and breast cancer AS-based biomarkers identified to date, leading to several high-impact publications.
• Ongoing collaborations on AS-related projects with over 20 laboratories worldwide.
• Deep sequencing, gene expression and AS analysis services provided to over 60 labs worldwide.
To date, 22 publications using data generated by this facility have appeared including these whose contributing authors are not directly associated with the platform including Besin et al., Eur J Immunol.42, 2491 (2012). Coulombe-Huntington et al., PLoS Genet 5, e1000766 (2009). Hai et al., Nucleic Acids Res 36, 3320 (2008). Montagne et al., PLoS One 7:e32172, (2012). Paquette et al., Br. J. Cancer 105, 534-41, 2011. Porquet et al., BMC Cancer11, 285, 2011. Warzecha et al., EMBO J 29, 3286 (2010). Warzecha et al., RNA Biol 6, 546 (2009). Zhang et al., Cell 133, 585 (2008).
Cloutier, A., Shkreta, L., Durand, M., Toutant, J., Thibault, P. and Chabot, B. (2018). hnRNP A1/A2 and Sam68 collaborate with SRSF10 to control the splicing response to oxaliplatin-mediated DNA damage. Sci. Rep. 8(1):2206. doi: 10.1038/s41598-018-20360-x. PMID: 29396485
Deshaies, J.E., Shkreta,L., Moszczynski, A.J., Flamier, A., Sidibe, H., Semmler, S., Fouillen, A., Bennet, E., Bekenstein, U., Destroismaisons, L., Toutant, J., Delmotte Q., Volkening, K., Stabile, S., Aulas, A., Khalfallah, Y., Soreq, H., Nanci, A., Bernier, G., Strong, M.J., Chabot, B. and Vande Velde, C. (2018). TDP-43 regulates hnRNP A1 alternative splicing to generate an aggregation prone isoform in amyotrophic lateral sclerosis. Brain March 19. doi: 10.1093/brain/awy062. PMID: 29562314
Bondy-Chorney, E., Baldwin, R.M., Didillon, A., Chabot, B., Jasmin, B.J. and Côté, J. (2017). RNA binding protein RALY promotes Protein Arginine Methyltransferase 1 alternatively spliced isoform v2 relative expression and metastatic potential in breast cancer cells. Int. J. Biochem. & Cell Biol. Jul 18. pii: S1357-2725(17)30172-3. doi:10.1016/j.biocel.2017.07.008 PMID: 28733251
Sutherland, L.C., Thibault, P., Durand, M., Lapointe, E., Knee J.M., Beauvais, A., Kalatskaya I., Hunt, S.C., Loiselle, J.J., Roy, J.G., Tessier, S.J., Ybazeta, G., Stein, L., Kotary, R., Klinck, R. and Chabot, B. (2017). Splicing arrays reveal novel RBM10 targets, including SMN2 pre-mRNA. BMC Mol. Biol. 18(1):19. doi: 10.1186/s12867-017-0096-x. PMID: 28728573
Deschênes, M. and Chabot, B. (2017). The emerging role of alternative splicing in senescence and aging. Aging Cell doi:10.1111/acel.12646. PMID: 28703483
Cloutier, P., Poitras, C., Durand, M., Hekmat, O., Fiola-Masson, E., Bouchard, A., Faubert, D., Chabot, B. and B. Coulombe. (2017). The RUVBL1/RUVBL2 component of the cochaperone R2TP/prefoldin-like directly interacts with ZNHIT2 to regulate assembly of the small nuclear ribonucleoprotein U5. Nature Commun. May 31;8:15615. doi: 10.1038/ncomms15615. PMID: 28561026.
Shkreta, L., Blanchette, M., Toutant, J., Wilhem, E., Bell, B., Story, B.A., Cochrane, A., Balachandran, A., Cheung, P.K., Harrigan, P.R., Grierson, D. and Chabot, B. (2017). Modulation of the splicing regulatory function of SRSF10 by a novel compound that impairs HIV-1 replication. Nucl. Acids Res. 45(7):4051-4067. doi: 10.1093/nar/gkw1223. PMID : 27928057.
Shkreta, L., Toutant, J., Durand, M., Manley, J.L. and Chabot, B. (2016). The splicing regulator SRSF10 connects the DNA damage response to the alternative splicing of genes encoding components involved in apoptosis, cell-cycle and DNA repair. Cell Reports 17(8):1990-2003. doi :10:1016/j.celrep.2016.10.071 PMID : 27851963.
Cheung, P.K., Horant, D., Bandy, L.E., Zamiri, M., Rabea, S.M., Karagiosov, S.K., Matloobi, M., McArthur, S., Harrigan, P.R., Chabot, B. and Grierson, D.S. (2016). A parallel synthesis approach to the identification of novel diheteroarylamide-based compounds blocking HIV replication: Potential inhibitors of HIV‐1 pre-mRNA alternative splicing. J. Med. Chem. 59(5):1869-79 doi:10.1021/ acs.jmedchem.5b01357 PMID: 26878150.
Bondy-Chorney, E., Crawford, T.E., Ravel-Chapuis, A., Klinck, R., Chabot, B., Jasmin, B.J., and Côté, J. (2016). Staufen1 regulates multiple alternative splicing events either positively or negatively in DM1 indicating its role as a disease modifier. PLoS Genet. 12(1): e1005827. doi:10.1371/journal.pgen.1005827 PMID : 26824521.
Chabot, B. and Shkreta, L. (2016). Defective control of pre-messenger RNA splicing in human disease. J. Cell. Biol. 212 (1):13-27. PMID: 26728853.
Shkreta, L. and Chabot, B. (2015). The RNA splicing response to DNA damage. Biomolecules 5(4): 2935-2977. PMID : 26529031.
Shkreta, L., Cloutier, A., Toutant, J., Vennin Rendos, H. and Chabot, B. (2015). Regulation of alternative splicing and the case of Bcl-x. Pak. J. Biochem. Mol. Biol. 48(2): 27-38.
Bergeron, D., Pal, G., Beaulieu, Y.B., Chabot, B. and Bachand, F. (2015). Regulated intron retention and nuclear pre-mRNA decay contribute to PABPN1 autoregulation. Mol. Cell. Biol. 35(14) 2503-17. PMID: 25963658.
Gabriel, M., Delforge, Y., Deward, A., Habraken, Y., Hennuy, B., Piette, J., Klinck, R., Chabot, B., Colige, A. and Lambert, C. (2015). Role of the splicing factor SRSF4 in cisplatin-induced modifications of pre-mRNA splicing and apoptosis. BMC Cancer. 15:227. doi: 10.1186/s12885-015-1259-0. PMID: 25884497.
Lemieux, B., Blanchette, M., Monette, A., Mouland, A.J., Wellinger, R.J. and Chabot, B. (2015). A function for hnRNP A1 and A2 in transcription elongation. PLoS One. 10(5):e0126654. doi: 10.1371. PMID: 26011126.
Chabot, B. (2015). My road to alternative splicing control: from simple paths to loops and interconnections. Biochem. Cell Biol. dx.doi.org/10.1139/bcb-2014-0161. PMID: 25759250.
Chabot, B. (2015). Finding the rules of splicing and using them….alternatively. RNA 21: 582-583(invited essay for the 20th anniversary commemorative issue of the RNA journal). PMID : 25780148.
Mauger, O., Klinck, R., Chabot, B., Muchardt, C., Allemand, E. and Batsché, E. (2015). Alternative splicing regulates the expression of G9A and SUV39H2 methyltransferases, and dramatically changes SUV39H2 functions. Nucl. Acids Res. 43:1869-1882 PMID: 25605796.
Best,A., James, K., Dalgliesh, C., Hong,E., Kheirolahi-Kouhestani, M., Curk, T., Xu, Y., Danilenko, M., Hussain, R., Keavney, B., Wipat, A., Klinck, R., Cowell, I., Lee, K. C., Austin, C., Venables, J., Chabot, B., Santibanez Koref, M., Tyson-Capper, A. and D. J. Elliott. (2014). Human Tra2 proteins jointly control a CHEK1 splicing switch among alternative and constitutive target exons. Nature Comm. 5:4760 PMID:25208576
Klinck, R., Fourrier, A., Thibault, P., Toutant, J., Durand, M., Lapointe, E., Caillet-Boudin, M.-L., Sergeant, N.,Gourdon, G., Meola, G., Furling, D., Puymirat, J. and Chabot, B. (2014). RBFOX1 cooperates with MBNL1 to control splicing in muscle, including events altered in myotonic dystrophy type 1. PLoS One 9(9):e107324. PMID: 25211016
Brosseau, J.P., Lucier, J.F., Nwilati, H., Thibault, P., Garneau, D., Durand, M., Couture, S., Lapointe, E., Prinos, P., Klinck, R., Perreault, J.P., Chabot, B. and Abou Elela, S. (2014). Tumor microenvironment associated modifications of alternative splicing. RNA. 20:189-201.
Brosseau, J.P., Lucier, J.F., Lamarche, A.A, Shkreta, L., Lapointe, E., Couture, S., Thibault, P., Gendron, D., Paquet, E., Perreault, J.P., Abou Elela, S. and Chabot, B. (2014). Redirecting splicing with bifunctional oligonucleotides. Nucl. Acids Res. 42(6):e40. Covered in Biotechniques. http://www.biotechniques.com/news/Snipping-and-Knitting-in-All-the-Right-Places/biotechniques-351323.html#.U1A488fxshl
Venables, J.P., Lapasset, L., Gadea, G., Fort, P., Klinck, R., Irimia, M., Vignal, E., Prinos,P., Chabot, B., Abou Elela, S., Roux, P., Lemaitre, J.M. and Tazi, J. (2013). MBNL1 and RBFOX2 cooperate to change a splicing program involved in pluripotent stem cell differentiation. Nature Comm. 4:2480 doi: 10.1038/ncomms3480.
Diaz, Z., Aguilar-Mahecha, A., Paquet, E.R., Basik, M., Orain, M., Camlioglu, E., Constantin, A., Benlimame, N., Bachvarov,, D., Jannot,, G., Simard, M.J., Chabot, B., Gologan, A., Klinck,R., Gagnon-Kugler, T., Lan,C., Przybytkowski, E., Qureshi, S., Rousseau,C., Spatz, A., Têtu,B., Batist, G. (2013). Next-generation biobanking of metastases to enable multidimensional molecular profiling in personalized medicine. Modern Pathology 26: 1413-1424.
Shkreta, L., Bell, B., Revil, T., Venables, J.P., Prinos, P., Abou Elela, S. and Chabot, B. (2013). Cancer-associated perturbations in alternative pre-messenger RNA splicing. Cancer Treat Res. 158: 41-94.
Venables, J.P., Brosseau, J.P., Gadea, G., Klinck, R., Prinos, P., Beaulieu, J.F., Lapointe, E., Durand, M., Thibault, P., Tremblay, K., Rousset, F., Tazi,J., Abou Elela, S. and Chabot, B. (2013). RBFOX2 is an important regulator of mesenchymal-specific splicing in both normal and cancer tissues. Mol. Cell. Biol. 33:396-405.
Prinos, P., Klinck, R., Venables, J.P., Perreault, J.P., Wellinger, R.J., Abou Elela, S. and Chabot, B. (2012). Alternative splicing as a generator of functional diversity in cancer. MSA 1(4):151-165 [In French]
Michelle, L., Barbier, J. and Chabot, B. (2012). hnRNP and hnRNP-like proteins in splicing control: molecular mechanisms and implication in human diseases. In RNA binding proteins, edited by Z. J. Lorkovic. Landes Biosciences. pp. 1-25.
Lund, N., Milev, M.P., Wong, R., Sanmuganantham, T., Woolaway, K., Chabot, B., Abou Elela, S., Mouland, A.J., Cochrane, A. (2012). Differential effects of hnRNP D/AUF1 isoforms on HIV-1 gene expression. Nucl. Acids Res. 40(8):3663-3675.
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., Abou Elela, S., Prinos, P. and Chabot, B.(2012). EJC components modulate the alternative splicing of Bcl-x and other apoptotic genes. Mol. Cell. Biol.32:954-967.
Montes, M., Cloutier, A., Sánchez-Hernández, N., Michelle, L., Lemieux, B., Blanchette, M., Hernández-Munain, C., Chabot, B. and Suñé, C. (2012). The elongation and splicing-related factor TCERG1 regulates alternative splicing of the Bcl-x apoptosis gene by modulating the rate of RNA polymerase transcription. Mol. Cell. Biol. 32:751-762.
Klinck, R., Chabot, B., Abou Elela, S. (2011) High-throughput RT-PCR. In "RNA splicing: The complete guide", edited by S. Stamm, C. Smith and R. Lührmann. Wiley-VCH.
Parenteau, J., Durand, M., Morin, G., Gagnon, J., Lucier, J.F., Welligner, R.J., Chabot, B. and Abou Elela, S. (2011). Introns regulate the production and functional specificity of yeast ribosomes. Cell 14:320-331.
Prinos, P., Garneau, D., Lucier, J.F., Gendron, D., Couture, S., Boivin, M., Brosseau, J.P., Lapointe, E., Thibault, P., Durand, M., Tremblay, K., Gervais-Bird, J., Nwilati, H., Klinck, R., Chabot, B., Perreault, J.P., Wellinger, R.J., Abou Elela, S. (2011). Alternative splicing of SYK regulates mitosis and cell survival. Nat. Struct. Mol. Biol. 18:673-679.
Tanackovic, G.,Ransijn, A., Thibault, P., Abou Elela, S., Klinck, R., Berson, E.L., Chabot, B. and Rivolta, C. (2011). Defects in pre-mRNA splicing and altered steady-state levels of spliceosome components are associated with retinal degeneration in humans. Hum. Mol. Genet. 20:2116-21130.
Shkreta, L., Michelle, L., Toutant, J., Tremblay, M.L. and Chabot, B. (2011). The DNA damage response pathway regulates the alternative splicing of the apoptotic mediator Bcl-x. J. Biol. Chem. 286:331-340.
Dominguez, C., Fisette, J.F., Chabot, B. and Allain, F.H.T. (2010). Structural basis of G-tract recognition and encaging by hnRNP F quasi-RRMs. Nat. Struct. Mol. Biol. 17:853-861.
Fisette, J.F., Toutant, J., Dugré-Brisson, S., Desgroseillers, L. and Chabot, B. (2010). hnRNP A1 and hnRNP H can cooperate to modulate 5’ splice site selection. RNA 16:228-238.