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L'épissage alternatif est un processus biologique qui génère la diversité du protéome malgré le nombre limité de gène. Ce mécanisme régule à la fois les gènes de manières qualitatives (isoformes exprimées) mais aussi quantitatives (niveau d'expression). Avec le développement des technologies de séquençage à haut débit, il est maintenant possible d'étudier à large échelle les aspects quantitatif et qualitatif du transcriptome avec une même expérience (RNA-seq). Durant ma thèse, j'ai développé une nouvelle méthode d'analyse de l'épissage alternatif dans les données RNA-seq. J'ai également participé à la mise en place du pipeline global d'analyse de données RNA-seq (expression et épissage) qui a été utilisé pour analyser un grand nombre de jeux de données. Dans un second temps, nous avons comparé notre outil d'analyse de l'épissage, FaRLine, qui est basé sur l'alignement sur un génome de référence, à KisSplice, une méthode basée sur l'assemblage. Nous avons montré que ces méthodes trouvaient un grand nombre d'événements en communs (70%), mais qu'il existait des différences non négligeables dues à la méthodologie. Nous avons analysé et classifié ces événements en 4 grandes catégories. Les variants faiblement exprimés et les exons chevauchant des éléments répétés sont mieux annotés par les méthodes basées sur l'alignement. Alors que les méthodes basées sur l'assemblage trouvent des nouveaux variants (exons ou sites d'épissage non annotés) et prédisent de l'épissage alternatif dans les famille de gènes paralogues. Notre travail souligne les points qui nécessitent encore l'amélioration des méthodes bioinformatiques. Enfin, j'ai participé au développement de méthodes permettant d'aider les biologistes à évaluer l'impact fonctionnel de modification d'épissage, que ce soit au niveau de la protéine produite (annotation des domaines protéiques au niveau des exons), ou à un niveau plus global en intégrant les modifications d'épissage dans les voies de signalisation / Alternative splicing is the biological process that explain the large diversity of the proteome compared to the limited number of genes. This process allow a qualitative regulation (expressed isoforms) and a quantitative regulation (expression level). The growth of high-trhoughtput sequencing methods enabled the analysis of these two aspects (quantitative and qualitative regulation) with the same experiment (RNA-Seq). During my PhD, I developped a new tool to analyse alternative splicing from RNA-Seq data. I also participated in the automatisation of the complet pipeline of RNA-Seq analysis (expression and splicing). This pipeline has been used to analyse various datasets. Then, we compared our mapping-first tool, FaRLine, with an assembly-first method, KisSplice. We found that the predictions of the two pipelines overlapped (70\% of exon skipping events were common), but with noticeable differences. The mapping-first approach allowed to find more lowly expressed splicing variants, and was better in predicting exons overlapping repeated elements. The assembly-first approach allowed to find more novel variants, including novel unannotated exons and splice sites. It also predicted AS in families of paralog genes. Our work point out where the bioinformatic improvment are still needed. Finally, I participated in the developpement of bioinformatics methods to help biologists to evualuate the fonctionnal impact of splicing alteration : at the level of the protein product by annotating fonctionnal domain at the exon level or at a more global level, by integrating splicing modifications in signaling pathways

The post-natal regenerative capacity of skeletal muscle is attributed to myogenic satellite cells, which function as lineage-committed precursors to replace terminally differentiated muscle. The development and differentiation of the satellite cell lineage is regulated by Pax7 and the myogenic regulatory factors. While the expression of Pax7 is vital to the function of the satellite cell compartment, the paired domain alternative splicing events that regulate its DNA binding potential remain elusive. Interestingly, the generation of Pax7 splice variants differentially regulate Myf5 expression. We performed a global analysis of two Pax7 isoforms, which differ by a glycine-leucine dipeptide, to determine how paired domain splicing events modify the ability of Pax7 to regulate target genes. To this end, we observe that although the homeodomain is important for Pax7 binding, these isoform differences in the paired domain can regulate Pax7 targets during myogenesis. In addition to further examining the role of Pax7 during satellite cell proliferation and maintenance, it remains important to understand their downstream differentiation potential. Since activation of the canonical Wnt signalling pathway results in reduced regenerative efficiency in vivo, we undertook a global analysis of satellite cell derived myoblasts to examine their ability to respond to canonical Wnt signalling. We demonstrate that Wnt/β-Catenin signalling drives myogenic differentiation, via the myogenin-dependent control of follistatin expression, further fine-tuning the myogenic differentiation process. The effects of canonical Wnt signalling on myogenic differentiation complement our observations regarding Pax7 alternative splicing during myoblast proliferation and provide a greater comprehensive understanding of the molecular regulation of satellite cell development and differentiation during adult myogenesis.

The field of proteogenomics operates at the interface between proteomics and genomics, and has emerged during the past decade to exploit the vast quantities of high-throughput sequence data. A range of different proteogenomics approaches have been developed, which integrate mass spectrometry data with genome sequence data to provide empirical evidence for protein-coding genes. However, current methods may not be optimized as they do not fully consider the splicing complexity in eukaryotes and there is currently no best practice method. To address this, we investigate the level of proteomics support for Ensembl gene models in human, and a selection of model organisms. We find a disparity between the number of splice variants confirmed by extant data, and the number that can theoretically be confirmed using current proteomics technologies. We then go on to investigate EST-based proteogenomics methods, which enabled the discovery of novel peptide sequences in the chicken genome, which represent hitherto unannotated genes, amended gene models, polymorphisms, and genes missing from the genome assembly. Different approaches for searching mass spectrometry data against transcript sequences are explored, and we show that searching mass spectra against protein sequences predicted by the EORF and ESTScan2 translation tools results in the best sensitivity.

Bioinformática estrutural de proteínas modificadas por eventos de splicing alternativo / Structural Bioinformatics of Proteins modified by Alternative Splicing

Type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of pancreatic β-cells, mediated by autoreactive T cells.The resulting inflammatory response takes place in the context of a dialogue between invading immune cells and the targeted β-cells, and it is modulated by genetic susceptibility, acting on both immune and β-cells, and by inflammatory cytokines and chemokines. Stress pathways triggered within β-cells may potentiate autoimmunity, and T1D susceptibility genes shape β-cell responses to “danger signals”, innate immunity, and activation of apoptosis. However, the molecular mechanisms linking genetic variation, environmental triggers, and the signaling events promoting β-cell dysfunction and loss remain poorly clarified. Pre-mRNA splicing is a crucial mechanism for gene expression regulation, and more than 95% of the human multi-exonic primary transcripts undergo alternative splicing. Splicing dysregulation have been increasingly recognized to play a pivotal role in multiple pathologies, including autoimmune diseases. More than 15% of the mutations described in the Human Gene Mutation Database are predicted to affect splicing. Our group has shown that exposure to pro-inflammatory cytokines induces major changes on the β-cell transcriptome, affecting the splicing of genes that are key for β-cell function and survival. Importantly, our group identified that GLIS3, a susceptibility gene for both T1D and type 2 diabetes (T2D), modulates β-cell apoptosis via regulation of the splicing factor SRSF6, linking T1D genetic susceptibility and alternative splicing. The downregulation of GLIS3, either by germline mutations associated with monogenic forms of diabetes or risk single nucleotide polymorphisms, contribute to SRSF6 splicing factor downregulation. Splicing factors are the primary regulators of splicing and orchestrate functionally related transcripts into regulatory networks, therefore, oscillations of splicing factors’ expression levels have a major impact on splicing decisions. In the present study we aimed: 1. To evaluate the functional impact of SRSF6 downregulation in human pancreatic β-cells; 2. To identify the SRSF6-regulated splicing networks and to decode the SRSF6 cis-regulatory RNA binding elements.To fulfil these aims, human insulin-producing EndoC-βH1 cells were subjected to RNA sequencing (under control conditions or following SRSF6 knock down for 48h) to identify transcriptome-wide alternative splicing events regulated by SRSF6, and to individual-nucleotide resolution UV crosslinking and immunoprecipitation followed by high-throughput sequencing (iCLIP) to determine the SRSF6 mechanistic model of splicing regulation, its associated cis-regulatory elements and directly bound transcripts in human β-cells. We observed that SRSF6 depletion has a major impact on human pancreatic β-cell function and survival, leading to β-cell apoptosis and impaired insulin secretion. SRSF6 downregulation affects the splicing of transcripts involved in central pathways for β-cell function and survival, such as insulin secretion (e.g. INSR, SNAP25), apoptotic regulators (e.g. BCL2L11 (or BIM), BAX), and the mitogen-activated protein kinases (MAPKs) signaling pathway (e.g. MAPK8, MAPK9, MAP3K7). SRSF6 silencing potentiates the generation of constitutively active isoforms of pro-apoptotic inducers – BAX-β, and BIM-Small - leading to apoptosis activation, and also of different members of the MAPK signaling pathway contributing to the hyper-phosphorylation of the pathway, leading to activation of down-stream transcription factors and consequent β-cell apoptosis. These data indicate that specific splicing networks, regulated through diabetes susceptibility genes, control key pathways and processes involved in the function and survival of β-cells. The iCLIP analysis has shown that SRSF6 recognizes more than 100,000 of RNA binding sites in protein coding sequences, and it regulates splicing by preferentially binding into exons through a purine-rich consensus motif consisting of GAA triplets. The number of triplets in direct sequence correlates with increasing binding site strength. The SRSF6 binding position affects the splicing outcome, possibly resulting from the competition between alternative exons and their flanking constitutive exons for SRSF6 tethering. We identified SRSF6 binding sites on SRSF6-regulated cassette exons of several susceptibility genes for both T1D and T2D, and as a proof-of-concept, modulated the splicing of the LMO7 susceptibility gene using antisense oligonucleotides.In conclusion, our data suggest that SRSF6 is a master splicing regulator in pancreatic β-cells, downstream of the diabetes susceptibility gene GLIS3. SRSF6 silencing potentiates the splicing of constitutively active pro-apoptotic variants (BAX-β and BIM-Small), and exacerbates the MAPK signalling pathway. SRSF6 recognizes specific purine-rich RNA binding motifs, with important implications for the interpretation of sequence variants. This work unveiled a novel regulatory layer for β-cell demise and diabetes genetic susceptibility, namely through splicing mis-regulation. These observations raise the possibility that splicing networks regulated by candidate genes for diabetes contribute to β-cell dysfunction and death in diabetes. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

La voie Hippo est une voie conservée impliquée dans la croissance des tissus et la suppression de tumeurs. Des études ont démontré son implication dans le développement des cancers chez l'homme. Cette cascade contrôle l'activité du co-activateur transcriptionnel Yorkie (Yki) chez la drosophile et de la protéine YAP (Yes Associated Protein) chez les mammifères. En raison de l'épissage alternatif de leur transcrits, les protéines Yki et YAP existent sous deux isoformes contenant un domaine WW (Yki1/YAP1) ou deux (Yki2/YAP2). Puisque les domaines WW sont essentiels pour l’interaction avec des partenaires spécifiques, l’inclusion alternative de ce domaine dans la protéine Yki/YAP peut remodeler leur réseau d’interaction et donc leur activité. La régulation et les conséquences fonctionnelles de l’épissage alternatif de yki / YAP in vivo sont inconnues.Dans le cadre de ce doctorat, nous avons constaté que la déplétion du facteur d’épissage B52 chez la drosophile réduit l’inclusion de l’exon alternatif dans l’ARNm de yki et favorise l’expression de l’isoforme Yki1 aux dépens de l’isoforme Yki2. La déplétion en B52 dans l'aile réduit la croissance et l'activité de Yki. Nous montrons que l'isoforme Yki1 est une version atténuée de la protéine Yki qui peut entrer en concurrence avec l'isoforme Yki2 dans le noyau. Pour déterminer le rôle de l’épissage alternatif de yki in vivo et l'importance de l'isoforme courte Yki1, nous avons abrogé cet épissage en utilisant la technologie CRISPR/Cas9 et avons créé des mouches capables d'exprimer uniquement l'isoforme Yki2. Ces mouches yki2only sont viables mais présentent un phénotype aléatoire d’ailes asymétriques. Cette augmentation de l'«asymétrie fluctuante», qui traduit une déviation par rapport au développement normal, suggère que l’épissage alternatif de yki est crucial pour la stabilité développementale. Ces résultats mettent en évidence un nouveau niveau de modulation de la voie Hippo via l’épissage alternatif de yki.L'inclusion alternative du deuxième domaine WW est une caractéristique conservée entre Yki et YAP. Cela conforte l'idée que les isoformes Yki1 et YAP1 ont une fonction importante in vivo et que l'épissage alternatif de yki/YAP est un mécanisme conservé de contrôle de la voie Hippo. Cette étude ouvre de nouvelles perspectives pour la modulation de la voie Hippo dans les cellules cancéreuses en modifiant l’épissage alternatif de YAP. / The Hippo pathway is a conserved pathway involved in tissue growth and tumor suppression. Studies have demonstrated its significance in the development of human cancers. This cascade controls the activity of the transcription co-activator Yorkie (Yki) in flies and Yes-associated protein (YAP) in mammals. Due to Alternative Splicing (AS), both Yki and YAP proteins exist as two isoforms containing one (Yki1/YAP1) or two (Yki2/YAP2) WW domains. Since WW domains are essential for interaction with specific partners, the alternative inclusion of this domain in Yki/YAP protein may remodel their interaction network and therefore their activity. The regulation and functional consequences of AS of yki/YAP in vivo are unknown.In this Ph.D. project, we identified that depletion of splicing factor B52 in Drosophila lowers inclusion of the alternative exon in yki mRNAs and favors the expression of Yki1 isoform at the expense of the Yki2 isoform. B52 depletion in the wing reduces growth and Yki activity. We demonstrate that Yki1 isoform is an attenuated version of Yki protein that can compete with Yki2 isoform in the nucleus. To ascertain the role of yki AS in vivo and the importance of short isoform Yki1, we abrogated this splicing by using CRISPR/Cas9 technology and created flies that can express Yki2 isoform only. yki2only flies are viable but display a random phenotype of asymmetric wing size. This rise in “fluctuating asymmetry” that is the consequence of subtle deviation from normal development, suggests that AS of yki is crucial for the development robustness. Taking together, these results highlight a new layer of modulation of Hippo pathway via AS of yki.Alternative inclusion of the second WW domain is a conserved feature between Yki and YAP. This further supports the idea that Yki1 and YAP1 isoforms have an important function in vivo and that AS of yki/YAP is a conserved mechanism of control of the Hippo pathway. This study opens up new perspectives for modulation of the Hippo pathway in cancer cells by altering YAP AS.

Both genetic and environment factors influence susceptibility to substance use disorders. However, the genetic basis of these disorders is largely unknown. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene for reduced methamphetamine (MA) stimulant sensitivity. Mutation (heterozygous deletion of a small region in the first coding exon) in Hnrnph1 also decreased MA reinforcement, reward, and dopamine release. 5’UTR genetic variants in Hnrnph1 support reduced 5’UTR usage and hnRNP H protein expression as a molecular mechanism underlying the reduced MA-induced psychostimulant response. Interestingly, Hnrnph1 mutant mice show a two-fold increase in hnRNP H protein in the striatal synaptosome with no change in whole tissue level. Proteome profiling of the synaptosome identified an increase in mitochondrial complex I and V proteins that rapidly decreased with MA in Hnrnph1 mutants. In contrast, the much lower level of basal mitochondrial proteins in the wild-type mice showed a rapid, MA-induced increase. Altered mitochondrial proteins associated with the Hnrnph1 mutation may contribute to reductions in MA behaviors. hnRNP H1 is an abundant RNA-binding protein in the brain, involved in all aspect of post-transcriptional regulation. We examined both baseline and MA-induced changes in hnRNP H-RNA interactions to identify targets of hnRNP H that could comprise the neurobiological mechanisms of cellular adaptations occurring following MA exposure. hnRNP H post-transcriptionally regulates a set of mRNA transcripts in the striatum involved in psychostimulant-induced synaptic plasticity. MA treatment induced opposite changes in binding of hnRNP H to these mRNA transcripts between Hnrnph1 mutants versus wild-types. RNA-binding, transcriptome, and spliceome analyses triangulated on hnRNP H binding to the 3’UTR of Cacna2d2, an upregulation of Cacna2d2 transcript, and decreased 3’UTR usage of Cacna2d2 in response to MA in the Hnrnph1 mutants. Cacna2d2 codes for a presynaptic, voltage-gated calcium channel subunit that could plausibly regulate MA-induced dopamine release and behavior. The multi-omics datasets point to a dysregulation of mitochondrial function and interrelated calcium signaling as potential mechanisms underlying MA-induced dopamine release and behavior in Hnrnph1 mutants.

Alternative splicing is a major contributing factor to protein diversity. The human genome project validated that there are about 25,000 genes, but over 100,000 proteins. Genes contain numerous exons that are specifically regulated; thus, elucidating alternative splicing mechanisms of pre-mRNAs is an immense undertaking. Two basic tools are used to study this mammoth task. For in vitro studies, usually in vitro transcription followed by splicing assays is used. For in vivo studies, the main technique is the construction of minigenes. These techniques enable one to study the mechanisms/conditions that explain an exon's inclusion or exclusion in the mature mRNA. In this project, studies were focused in the protein kinase C (PKC) family, and specifically in certain exons of PKCbeta and PKCdelta genes. The PKCbeta gene codes two proteins: PKCbetaI and PKCbetaII. The pre-mRNA of PKCbeta contains 18 exons. If exon-17 (exon-betaII) is included in the spliced transcript, protein PKCbetaII is expressed. If exon-betaII is skipped (excluded), protein PKCbetaI is expressed. Previous and current results indicate that insulin treatment not only favors exon-betaII inclusion, but also regulates 3?-UTR size in mature mRNA. We identified up to five different PKCbetaII mRNAs that differ only in the 3?-UTR size (all five mature transcripts express the same PKCbetaII protein). PKCbetaII is required for glucose uptake in skeletal muscle and adipocytes. Additional studies revealed that inclusion of exon-betaII involves SRp40 in its phosphorylated form. Small interfering RNA (siRNA) knockdown of Akt2 and Clk1 further revealed that SRp40 was phosphorylated by Akt2/Clk1, and that Akt2 phosphorylates Clk1. The knockdown of Clk1 or SRp40 down-regulated glucose uptake. The PKCdelta gene contains 18 exons. The standard size of exon-10 is 97-bases, and when included in the mature transcript, PKCdeltaI is expressed. Exon-10 has an additional 5?-splice site that extends the exon size to 190-bases. When this extended size is included in the mature transcript, PKCdeltaVIII is expressed which has a profound effect in the fate of the cell by blocking apoptosis. Our results reveal that all-trans retinoic acid regulates the inclusion of exon-10 extended size. Moreover, the inclusion involves SC35 (SRp30b). Finally, we elucidated the position of the SC35 cis-element.

Protein Prp22 is a DEAH box RNA helicase, which plays two distinct roles in pre-mRNA splicing: it participates in second transesterification step (ATP independent function) and it releases mature mRNA from the spliceosome (ATP dependent function). Prp45p, yeast ortholog of the human transcription co-regulator SNW/SKIP, is an essential splicing factor, it is included in spliceosome throughout the splicing reaction. Mutant prp45(1-169) genetically interacts with some alleles of NTC complex and second step splicing factors, one of them is also gene PRP22. Here we present, that mutants prp22(-158T) and prp22(-327A), which are synthetically lethal with prp45(1-169), express lower amount of Prp22p due to the mutation in upstream regulation region. Mutants prp22(-158T), prp22(300PPI) and prp22(-327A) affect splicing of pre-mRNA with mutation in 5'ss with respect to sequence of the second exon. N-terminal mutants prp22(∆301) and prp22(∆350) are synthetically lethal with prp45(1-169). Synthetic lethality is possibly caused by lower efficiency of Prp22 recruitment to the spliceosomes, which is no more viable for cells.