CHABOT Benoit, Ph.D. Professeur titulaire M. Sc. (Microbiologie) Université de Sherbrooke, Sherbrooke, Québec (1981) Ph. D. (Molecular Biophysics and Biochemistry) Yale University. New Haven, CT, USA. (1986) Formation post-doctorale Mount Sinai Hospital Research Institute, Toronto, Ontario (1986-88) Benoit.Chabot@USherbrooke.ca Chaire de recherche du Canada en génomique fonctionnelle/Canada Research Chair in Functional Genomics

English version

ÉPISSAGE ALTERNATIF DES ARNS PRÉMESSAGERS 

L'ÉPISSAGE ALTERNATIF

L'épissage alternatif des ARNs pré-messagers représente une stratégie importante permettant de moduler l'expression des gènes de façon spatio-temporelle chez tous les organismes supérieurs. Ce processus permet à certains exons, ou portions d'exons, d'être inclus de façon variable, mais précise dans les ARNs messagers, permettant ainsi la synthèse d'isoformes protéiques étalant des différences fonctionnelles. La régulation de l'épissage alternatif fait partie du programme d'expression d'une grande variété de gènes et a des conséquences importantes sur la physiologie et le développement animal, de même que sur l'apparition et l'évolution des maladies. La nature essentielle de ce processus biologique est illustrée par l'observation que les mécanismes de contrôle de l'épissage alternatif sont bouleversés dans les tissus cancéreux. Chez les gènes impliqués dans l'apoptose, l'épissage alternatif contrôle la production d'ARNm codant pour des protéines pro-apoptotiques ou anti-apoptotiques. Reconnaissant l'importance de ce processus, nos études sont principalement axées sur le gène qui code pour les protéines hnRNP A1 et sur le gène apoptotique Bcl-x.

MÉCANISMES DE L'ÉPISSAGE ALTERNATIF DE hnRNP A1

Les protéines hnRNP A1 sont des régulateurs de l'épissage alternatif et cette étude permet d'aborder les mécanismes impliqués dans l'autorégulation de l'épissage alternatif, un concept qui n'a jusqu'à maintenant pas encore été examiné chez les mammifères. Nos travaux ont permis d'identifier plusieurs éléments conservés dans les introns flanquant l'exon alternatif du gène A1. La protéine A1 est requise pour l'activité d'au moins deux de ces éléments. Des pré-mRNAs modèles contenant diverses combinaisons d'éléments conservés servent à examiner le mode d'action de ces éléments dans la sélection des sites d'épissage in vitro et in vivo. Le mécanisme par lequel hnRNP A1 influence l'interaction des facteurs d'épissage constitutif et coordonne l'action des éléments conservés aidera à mieux comprendre comment l'autorégulation de l'épissage alternatif fonctionne et permettra d'identifier d'autres éléments introniques conservés qui agissent sur la sélection des sites d'épissage 3'.  Ce système permet d'étudier la coordination entre une variété d'éléments qui modulent la sélection des sites d'épissage.

ÉPISSAGE ALTERNATIF DE GÈNES APOPTOTIQUES

Les mécanismes de contrôle de l’épissage alternatif sont souvent bouleversés dans les tissus cancéreux.  Chez les gènes impliqués dans l’apoptose (e.g., récepteur Fas, Mcl-1, CC3, Bcl-x et caspases), l’épissage alternatif contrôle la production d’ARNm codant pour des protéines pro-apoptotiques ou anti-apoptotiques.  Comme les processus normaux de contrôle sont perturbés dans les cellules transformées et que seules les formes anti-apoptotiques sont produites, nos activités de recherche étudient comment s'établit la sélection des sites d'épissage menant à la production des formes pro-apoptotiques et anti-apoptotiques.

L'approche principale est centrée sur Bcl-x qui produit par épissage alternatif deux protéines distinctes: la forme anti-apoptotique Bcl-xL et la forme pro-apoptotique Bcl-xS.  Afin d’identifier les éléments sur l’ARN pré-messager impliqués dans le contrôle de la sélection des sites et comprendre leurs mécanismes d’action, des approches in vivo et in vitro sont utilisées.  Des mini-gènes et des ARNs pré-messagers modèles portant des délétions d’exons ou d’introns ou contenant des éléments potentiellement impliqués dans le contrôle de l’épissage sont utilisés.  Cette approche permettent de vérifier l’activité de ces éléments et d’initier la caractérisation des facteurs qui médient ces activités.  La découverte des mécanismes utilisés par Bcl-x pour contrôler la sélection des sites d’épissage sera probablement pertinente aux modes de contrôle utilisés par d’autres gènes apoptotiques et permettra de solidifier notre compréhension du processus de l’épissage alternatif.

En collaboration avec le Dr Sherif Abou Elela, nous examinerons la contribution d'un grand nombre de protéines liant l'ARN à l'épissage alternatif de plusieurs centaines de gènes impliqués dans le cancer. Cette approche génomique utilisant l'interférence à l'ARN permettra de révéler l'organisation du contrôle de l'épissage alternatif et les réseaux moléculaires qui les régissent.

Finalement, en collaboration avec le Dr Raymund Wellinger, nous étudions le rôle des protéines hnRNP A1 et A2 dans la biogénèse des télomères.

ALTERNATIVE SPLICING OF ALTERNATIVE PRE-MESSENGER RNA IN MAMMALIAN CELLS

Following the completion of sequencing projects for several eucaryotic genomes (yeast, Drosophila, human), one of the outstanding challenges facing scientists around the world will be to determine the function of all genes and to understand how their expression is coordinated during cell growth and throughout development. It is noteworthy that the protein-coding portion of each mammalian gene represents only a small portion (on average less than 5%) of the total gene sequences, non-coding intron sequences representing the remaining 95%. Thus, even if we knew the function of each gene/protein, on a genome scale we would still do not know the function of the vast majority of the sequences. A large fraction of the genome is made-up of non-coding segments (introns) separating coding portions of genes (exons). Introns must be removed precisely and efficiently to insure proper synthesis of mRNA. Because mammalian introns can be very large (on average several kilobases to more than 200 Kb), and because the sequence of mammalian splicing signals is not highly conserved, one challenging endeavor is to understand how the splicing machinery selects the correct pair of splice sites. This process is starting to be understood for small introns in simple splicing units. The problem remains formidable for large introns. Alternative pre-mRNA splicing involves the differential use of splice sites, a process that represents a powerful and versatile way to control gene expression and, hence, protein function. Moreover, splice site selection can be regulated in a developmental, cellular, tissue and sex-specific manner. Recent studies have revealed the importance of alternative splicing in the expression strategies of complex organisms. It is estimated that probably more than 74% of all the human genes are alternatively spliced. Since many genes have more than two and some potentially up to several thousand alternatively spliced mRNA isoforms, the identity of more than 95% of the total number of human proteins may be determined by alternative pre-mRNA splicing. Despite the recognition of alternative pre-mRNA splicing as a critical process during vertebrate development and as a generator of protein diversity, very little is known about the identity of modulating factors and the underlying molecular mechanisms. In most cases, the nature of regulatory elements, the identity of trans-acting factors and the mechanisms of regulation remain unknown.

hnRNP A1 as a model to understand alternative splicing. Because of the paucity of information regarding the molecular mechanisms of alternative splicing, we have initiated investigations on the hnRNP A1 pre-mRNA which is alternatively spliced to yield the A1 and A1b mRNAs. The A1 protein is very abundant in the nucleus of actively growing mammalian cells and has been implicated in splice site selection, RNA packaging, RNA transport and telomere biogenesis. How A1 and A1b modulate splice site selection remains to be understood. It is a major objective of our current research to understand the molecular mechanisms by which hnRNP A1 affects splice site selection. We are characterizing sequences and factors involved in the alternative splicing of the A1 pre-mRNA with the aim of understanding how these elements communicate with each other and with the splicing machinery to modulate splice site selection. Our research objectives are therefore to understand how alternative splicing of the A1 pre-mRNA is coordinate with special attention to the contribution of the A1 proteins in the alternative splicing of its own pre-mRNA. With support from the CIHR, we have recently identified several elements in the introns flanking the alternative exon in the hnRNP A1 pre-mRNA. One element forms a highly stable secondary structure that sequesters the 5' splice site of the alternative exon and interferes with its recognition. Two elements located on each side of the alternative exon are essential for maximal exon skipping in vivo and distal 5' splice site selection in vitro. Both elements contain a high-affinity binding site for A1, and A1 binding to these elements is required for activity. We have noted the higher prevalence of putative A1 binding sites in introns, particularly near splicing signals. This observation raises the possibility that the model proposed to explain the mechanism of action of A1 in alternative splicing is also applicable to the splicing of large introns. Thus, we are also actively testing the relevance and contribution of A1 binding sites to the definition of large introns. We have also uncovered elements that repress 3' splice site utilization. We have identified an element that represses the 3' splice site of the common exon, to improve the inclusion of the alternative exon. We have used RNA chromatography to identify SRp30c as the binding factor. Thus, we are addressing the mechanisms governing the alternative splicing of a splicing regulator. Important insights will continue to be gained by studying the parameters that contribute to the alternative splicing of hnRNP A1. Already, we have identified several novel elements that affect splice site selection, as well as demonstrating a role for A1 and A1b in modulating he splicing of its own pre-mRNA. The hnRNP A1 system offers the advantage of studying the interplay between a variety of elements including secondary structure and distinct positive and negative elements, some of them bound by SR, other by hnRNP A1 proteins. We are convinced that understanding how splice site selection in the A1 pre-mRNA is coordinated will be relevant to other alternative splicing units and will help identify other target pre-mRNAs that also regulated by hnRNP A1 proteins.

Alternative splicing of apoptotic genes

Misregulation of alternative splicing is a common characteristic of human cancer.  In genes implicated in apoptosis (e.g., Fas receptor, Mcl-1, CC3, Bcl-x and caspases), alternative splicing controls the production of mRNAs encoding either pro-apoptotic or anti-apoptotic proteins.  Because the normal control of the apoptotic process is often perturbed in transformed cells, approaches that are aimed at (1) understanding how the production of pro-apoptotic and anti-apoptotic forms is regulated, and (2) uncovering new ways to interfere with splicing control, are intimately relevant to cancer.

The Bcl-x gene produces two spliced isoforms: the anti-apoptotic Bcl-xL protein and the pro-apoptotic Bcl-xS variant.  To uncover RNA elements involved in splicing control, and to investigate their mechanism of action, we are using a combination of in vivo and in vitro approaches. Mini-genes and model pre-mRNAs lacking portions of exons/introns or containing distinct putative control elements are used to identify and confirm the activity of elements, and to determine the identity of the trans-acting factor(s) involved in splicing modulation.  Uncovering the mechanisms used by Bcl-x to control the selection of splice sites will provide much needed insights into this crucial but poorly understood process.   

Publications récentes

Fisette, J.F., Toutant, J., Dugré-Brisson, S., DesGroseillers, L. and Chabot, B. (2010). hnRNP A1 and hnRNP H can cooperate to medulate 5' splice site selection. RNA 16, 228-238.

Alló, M. Buggiano, V., Fededa, J.P., Petrillo, E., Schor, I., de la Mata, M., Plass, M., Agirre, E., Eyras, E., Abou Elela, S., Klinck, R., Chabot, B. and Kornblihtt, A.R. (2009). Control of alternative splicing through siRNA-mediated transcriptional gene silencing. Nat. Struct. Mol. Biol. 16, 717-724.

Revil, T., Pelletier, J., Toutant, J., Cloutier, A., and Chabot, B. (2009). Heterogeneous nuclear ribonucleoprotein K represses the production of the pro-apoptotic Bcl-xS splice isoform. J. Biol. Chem. 284, 21458-21467.

Venables, J.P., Klinck, R., Koh, C., Bramard, A., Inkel, L., G.,  Gervais-Bird, J., Durand, M., Couture, S., Froehlich, U., Lapointe, E., Lucier, J.F., Thibault, P., Rancourt, C., Tremblay, K., Prinos, P., Chabot, B. and Abou Elela, S. (2009).  Cancer-associated alternative splicing. Nat. Struct. Mol. Biol. 16, 670-676.

Fisette, J.-F., Michelle, L., Revil, T. and Chabot B. (2009). Guiding and integrating to control and diversify splicing. Med. Sci. (Paris) 25(2), 175-780. In French.

Chabot, B., Abou Elela, S. and Zhuo, D. (2008). Comment on "When good transcripts go bad: artifactual RT-PCR 'splicing' and genome analysis". BioEssays 30, 1256.

Venables, J.P., Klinck, R., Bramard, A., Inkel, L., Dufresne-Martin, G., Koh, C., Gervais-Bird, J., Lapointe, E., Gendron, D., Brosseau, J.P., Thibault, P., Lucier, J.-F., Tremblay, K., Prinos, P., Wellinger, R., Chabot, B., Rancourt, C. and Abou Elela, S. (2008). Alternative splicing markers for breast cancer. Cancer Res. 68, 9525-9531.

Venables, J.P., Koh, C.S., Froehlich, U., Lapointe, E., Couture, S., Inkel, L., Bramard, A., Paquet, E.R., Watier, V., Durand, M., Lucier, J.-F., Gervais-Bird, J., Tremblay, K., Prinos, P., Klinck, R., Abou Elela, S. and Chabot, B. (2008). Multiple and specific alternative splicing targets for the major human hnRNP proteins. Mol. Cell. Biol. 28, 6033-6043.

Cloutier, P., Toutant, J., Shkreta, L., Goekjian, S., Revil, T. and Chabot B. (2008). Antogonistic effets of the SRp30c protein and cryptic 5'splice sites on the alternative splicing of the apoptotic regulator Bcl-x. J. Biol. Chem. 283, 21315-21324.

Shkreta, L., Froelich, U., Paquet, E.R., Toutant, J., Abou Elela, S. and Chabot, B. (2008). Impact of anticancer drugs on the alternative splicing of Bcl-x and other apoptotic genes. Mol. Cancer Ther. 7(6), 1398-1409.

Parenteau, J., Durand, M., Véronneau, S., Lacombe, A.-A. Morin, G., Guérin, V., Gervais-Bird, J., Koh, C.-S., Brunelle, D., Cecez, B., Wellinger, R., Chabot, B. and Abou Elela, S. (2008). Systematic deletion of many yeast introns reveals a minority of genes that require splicing for function. Mol. Biol. Cell. 19, 1932-1941.

Martinez-Contreras, R., Fisette, J. F., Cloutier. P., Revil, T., Shkreta L. and Chabot, B. (2007). hnRNP proteins and splicing control. Landes Biosciences Series. Alternative splicing in the post-genomic era. B. Blencowe and B. Graveley, editors. Adv. Exp. Med. Biol. 623, 123-147.

Revil, T., Toutant, J., Shkreta, L., Garneau, D., Cloutier, P. and Chabot, B. (2007). Protein kinase C-dependent control of Bcl-x alternative splicing. Mol. Cell. Biol. 27, 8431-8441.

Bakkour, N., Lin, Y. L., Maire, S., Ayadi, L., Mahuteau-Betzer, F., Nguyen, C. H., Mettling, C., Portales, P., Grierson, D., Chabot, B., Jeanteur, P., Branlant, C., Corbeau, P., Tazi, J. (2007). Small-molecule inhibition of HIV pre-mRNA splicing as a novel antiretroviral therapy to overcome drug resistance. PLoS Pathog. 3,1530-1539.

Jéronimo, C., Forget, D., Bouchard, A., Li, Q., Chua, G., Poitras, C., Thérien, C., Bergeron, D., Bourassa, S., Greenblatt, J., Chabot, B., Poirier, G., Hugues, T. R., Blanchette, M., Price, D. and Coulombe, B.(2007). Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme. Mol.  Cell 27, 262-274.

Paradis, C., Cloutier, P., Toutant, J., Klarskov, K., and Chabot B. (2007). hnRNP I/PTB can antagonize the splicing repressor activity of SRp30c. RNA 13, 1287-1300.

Zhuo, D., Madden, R., Abou Elela, S. and Chabot B. (2007). Modern origin of numerous alternatively spliced human introns from tandem arrays. Proc. Natl. Acad. Sci. U.S.A. 104, 882-886.

Revil, T., Shkreta, L., and Chabot, B. (2006). [Pre-mRNA alternative splicing in cancer: functional impact, molecular mechanisms and therapeutic perspectives] Bull. Cancer 93(9), 909-19. In French.

Gendron, D., Carriero, S., Garneau, D., Villemaire, J., Klinck, R., Abou Elela, S. Damha, M. J. and Chabot, B. (2006) Modulation of 5' splice site selection of the human Bcl-x pre-mRNA using tailed oligonucleotides carrying splicing signals. BMC Biotechnology 6, 5.

Martinez-Contreras, R., Fisette, J.-F., Nasim, F.H., Madden, R., Cordeau, M. and Chabot, B. (2006). Intronic binding sites for hnRNP A/B and hnRNP F/H proteins stimulate pre-mRNA splicing. PLoS Biol. 4, 172-185 (selected by BMC Faculty of 1000 Biology).

Naud, J.-F., McDuff, F.-O., Sauvé, S., Montagne, M., Webb, B.A, Smith, S.P., Chabot, B. and Lavigne, P. (2005). Structural and thermodynamical characterization of the complete p21 gene product of Max. Biochemistry 44, 12746-12758.

Garneau, D., Revil, T., Fisette, J.-F., Chabot, B. (2005). hnRNP F/H proteins control the alternative splicing of the apoptotic mediator Bcl-x. J. Biol. Chem. 280, 22641-22650.

Patry, C., Lemieux, B., Wellinger, R. and Chabot B.(2004). Targeting hnRNP A1 and A2 by RNA interference promotes cell death in transformed but not normal mouse cell lines. Mol. Cancer Ther. 3(10), 1193-1199.

Bériault, V., Clément, J.F., LeBel, C., Yong, X., Chabot, B., Cohen, E.A., Cochrane, A.W., Rigby, W.F.C. and Mouland, A.J. (2004). A late role for the association of hnRNP A2 with the HIV-1 hnRNP A2 response elements in genomic RNA, Gag and Vpr localization. J. Biol. Chem. 279, 44141-44153.

Villemaire, J., Dion, I., Abou Elela, S. and Chabot, B. (2003). Reprogramming alternative pre-messenger RNA splicing through the use of protein-binding antisense oligonucleotides. J. Biol. Chem. 278, 50031-50039.

Patry, C., Bouchard, L., Labrecque, P., Gendron, D., Lemieux, B., Toutant, J., Lapointe, E., Wellinger, R. and Chabot, B. (2003). siRNA-mediated reduction in hnRNP A1/A2 proteins induces apoptosis in human cancer cells but not in normal mortal cell lines. Cancer Res. 63, 7679-7688.

Gagné, J.P., Hunter, J., Labrecque, B., Chabot, B., and Poirier, G. (2003). A proteomic approach to the identification of heterogeneous nuclear ribonucleo-proteins as a family of poly(ADP-ribose)-binding proteins. Biochem., J. 371, 331-340.

Naud, J.-F., Gagnon, F., Chabot, B., Wellinger, R., Lavigne, P. (2003). Improving the thermodynamic stability of the leucine zipper of max increases the stability of its b-HLH-LZ:E-box complex. J. Mol. Biol. 326, 1577-1595.

Chabot, B., LeBel, C., Hutchison, S., Nasim, F.H. and Simard, M.J. (2003). HnRNP A/B proteins and the control the alternative splicing of the mammalian hnRNP A1 pre-mRNA. Progress in Molecular and Subcellular Biology. Regulation of alternative splicing. Springer-Verlag GmbH & Co. Heidelberg. Vol. 31, pp. 59-88.

Renseignements

Benoit Chabot, Ph.D.
Professeur titulaire
Département de microbiologie et d'infectiologie
Chaire du Canada en génomique fonctionnelle
Faculté de médecine et des sciences de la santé
Université de Sherbrooke
3001, 12e Avenue Nord
Sherbrooke (Québec) J1H 5N4
Téléphone: (819) 564-5295
Télécopieur: (819) 564-5392
Courrier électronique: Benoit.Chabot@USherbrooke.ca

RIBO-CLUB Sherbrooke : http://www.riboclub.org
Chaires de recherche du Canada : http://www.chairs.gc.ca

Le laboratoire de génomique fonctionnelle de l'Université de Sherbrooke (LGFUS) : http://lgfus.ca/public/

Le Centre de Recherche Clinique Étienne-Lebel : http://www.crc.chus.qc.ca/index.php