Exonic sequences of eukaryotic genes are interspersed with introns which when accurately removed from the primary transcript (pre-mRNA) results in a functional transcript. These splicing reactions are carried out by the spliceosome, consisting of U1, U2, U4, U5, U6 snRNAs and 150 non-snRNP proteins, which assemble onto the pre-mRNA and catalyzes the two invariant transesterification reactions (Will and Luhrmann, 2006). The flexibility in choice of splice sites allows for alternative splicing which has immensely contributed to eukaryotic genome evolution and in diversifying the metazoan proteome (Nilesen and Graveley, 2010). Dynamic yet ordered interactions between U2, U5 and U6 snRNAs and Prp8, Prp16, Prp17, Prp18, Slu7 and Prp22 splicing factors are required in vitro for second-step of splicing of budding yeast and human model transcripts (Umen and Guthrie, 1995a; Horowitz, 2012). ScSlu7 aids 3’ss selection while its strongly associated partner ScPrp18 stabilises U5 snRNA-exonic interactions (James et al., 2002; Aronova et al., 2007). These factors are dispensable in vitro, for the splicing of introns with short branch nucleotide to 3’ss distances (Brys and Schwer, 1996; Zhang and Schwer, 1997). Nearly 43% of fission yeast genes have short introns, with degenerate splice-signals and unconventional Py(n) tracts (Kuhn and Kaufer, 2003). As these features differ extensively from budding yeast and are interestingly more representative of fungal and other eukaryotic introns, fission yeast is an attractive unicellular model to investigate alternate splice-site recognition and assembly mechanisms. Mechanistic details of the second catalytic step are poorly understood in fission yeast. Strikingly, mutations in 3’ss and Py(n) tract intronic cis elements, known to block second step splicing in budding yeast, cause pre-catalytic arrest with unspliced pre-mRNA accumulation in fission yeast (Romfo and Wise, 1997). Studies in our laboratory focussed on understanding the functions for fission yeast SpPrp18 and SpSlu7 predicted to be second-step factors, revealed remarkable differences as compared to their budding yeast counterparts. Unexpectedly, SpPrp18 and SpSlu7 were found by our lab to be required before catalysis and these proteins do not directly associate with each other. Genome-wide splicing studies in a missense slu7-2 mutant indicated widespread yet intron-specific splicing functions for SpSlu7 (Banerjee et al., 2013). Crucial functions were attributed to helix-5 and conserved region loop of SpPrp18 and in vivo splicing analysis in selected cellular transcripts in a missense mutant (V194R) also revealed intron-specific functions (Thesis, N Vijaykrishna). In this study, we have advanced our understanding of SpPrp18 functions by identifying its global substrates and correlating with its intron-specific roles. Through molecular and genetic approaches, we have probed its role in splicing/spliceosome assembly. We identified intronic features within substrates that increase the propensity for the requirement of SpSlu7 for efficient splicing. Further, using findings from the genome-wide alternative splicing patterns in SpSlu7 and SpPrp18 mutants, we have attempted to understand their role in splice-site choice and thus alternative splicing. Ia. Understanding global splicing functions and spliceosomal interactions of fission yeast splicing factor SpPrp18 Since SpPrp18 is an essential gene, our lab generated the strains (prp18-5int [V194R] and WTint), where the thiamine-repressible promoter allowed conditional expression of wild-type or mutant allele integrated at the heterologous leu1 locus. Splicing efficiency of certain cellular transcripts with differing intron characteristics was assessed by semi-quantitative RT-PCR studies and the data suggested intron-specific SpPrp18 roles (in collaboration with Vijaykrishna N). This prompted us to investigate the global splicing role for SpPrp18 for which we used splicing-sensitive microarrays having custom-designed probes to distinguish unspliced pre-mRNA and spliced mRNA for every individual pombe intron. RNA from prp18-5int (V194R) and WTint cells was used in these experiments. We derived a stringent dataset of 258 introns which were statistically significant and correlated in two biological replicate RNA samples, for various probes. Hierarchical clustering of this dataset showed that the depletion of wild-type SpPrp18 triggered a range of splicing phenotypes like (A) pre-mRNA accumulation with mRNA reduction (B) pre-mRNA accumulation (C) spliced mRNA reduction and (D) unchanged pre-mRNA and mRNA levels. Statistical analysis of cis motifs that may correlate with the substrate-specific SpPrp18 splicing functions was done, but the data showed a lack of a global discriminatory primary sequence feature. However, a subtle intron-specific role for Py(n) tracts located between 5’ss and BrP was deduced for SpPrp18. This lead was validated by examining the in vivo splicing efficiency of minitranscripts with wild-type or an altered Py tract length, carried out for a SpPrp18 dependent and an independent intron. To specifically address if SpPrp18 activity was required for second-step splicing we investigated, using primer extension analyses, for lariat intron-3’exon species, an intermediate formed after step 1. We observed that even in prp18-5int dbr1∆ double mutants (where lariat molecules are not degraded) the cells accumulate only unspliced pre-mRNA and not lariat intermediates, a signature of an early arrest prior to the first transesterification reaction. Strengthening these findings, positive genetic interactions were noted between prp18-5int and ts mutants in two factors (U2AF59 and SpPrp1) involved in precatalytic spliceosome assembly and activation. On the whole, our genome-wide studies indicate intron-specific pre-catalytic functions for SpPrp18 supported by genetic interactions with early acting splicing factors involved in spliceosomal assembly and activation. Ib. Identification of intronic features that determine substrate-specific splicing functions for SpSlu7 In vitro studies with ScSlu7 and hSlu7 show their influence in 3’ss selection when BrP to 3’ss distance is greater than 7 nts and 23 nts respectively; but the global substrates are not known in either species (Brys and Schwer, 1996; Chua and Reed, 1999b). Genome-wide analysis of the splicing efficiency changes in cells with the mis-sense spslu7+ mutant (slu7-2), previously carried out in our lab, revealed a spectrum of splicing defects (Banerjee et al., 2013). To further understand the intron context-specific roles for SpSlu7, we examined intronic cis features that may correlate with SpSlu7 dependence. Statistical analyses of the affected (422 introns) and unaffected categories (90 introns) revealed that intron length, BrP to 3’ss distance and AU content are multiple discriminatory cis features that govern SpSlu7 splicing functions. To assess the contribution of these intronic features we tested whether altering these cis elements changes a transcript’s dependency (or otherwise) on SpSlu7 by RT-PCR analyses. For these studies, we generated plasmid expressed mini-genes containing the respective wild-type intron or intron with altered BrP-3’ss distances. We used nab2+ I2 as a case of an intron spliced independent of SpSlu7 and rhb1+ I1 as a representative for SpSlu7 dependent intron. Experiments testing their in vivo splicing status proved that BrP-3’ss distance is a cis feature that dictates SpSlu7 splicing functions in a context-dependent manner. The intronic AU content particularly between the 5’ss and the BrP was assessed in minigene constructs where a chimeric intron was generated by swapping the low AU containing sequences in the 5’ss to BrP stretch of cdc2+ I2 with AU rich bpb1+ I1 5’ end sequences. The results reaffirmed that low intronic AU content particularly at the 5’ end co-relates with SpSlu7 dependency. Hence, we have deduced novel intronic elements, which perhaps in combination, create a contextual dependence for SpSlu7 to facilitate efficient splicing. II. Alternative splice-site selection in fission yeast and studies on the role of splicing factors SpSlu7 and SpPrp18 Budding yeast second-step splicing factors ScSlu7 and ScPrp18 mediate 3’ss choice in the single intron containing transcripts. Fission yeast genome encodes cis and trans factors that promote alternative splicing similar to higher eukaryotes. In this study, we have devised a data analysis pipeline to identify alternative splice events in multi-intronic transcripts of fission yeast. Further, we utilised this information to interrogate the global role for SpSlu7 and SpPrp18 in alternate splice site selection. We mapped the microarray probe sequences corresponding to all theoretically possible non-consecutive splice junctions of S. pombe transcripts onto two independent experimental next-generation (NGS) transcriptomes from wild-type samples and identified 104 exon skipping events with NGS reads more than 3 (Wilhelm et al., 2008; Rhind et al., 2011). We further generated a stringent list of ten exon skipping events having high sequence reads as well as raw intensity value in our microarray experiments with wild-type cells. Two representative events from this list, an abundant rps13+ exon 2 skipped alternative mRNA and less abundant ats1+ exon 3 skipped alternative mRNA were then taken up for experimental analyses by semi-quantitative RT-PCR assays. We confirmed these events and further noted that SpSlu7 and SpPrp18 were required for the constitutive splicing of ats1+ E2-I2-E3-I3-E4 cassette. On the other hand, SpSlu7, and not SpPrp18, exerted a subtle influence on the skipping of exon 3. In addition to exon 3 skipped mRNA, we detected an intron 3 retained ats1+ alternative mRNA (E2-E3-I3-E4) in wild-type cells. Assessment of this event in cells metabolically depleted of SpSlu7 and SpPrp18 showed a reduced abundance of this species in both instances. This suggests a role for functional SpSlu7 and SpPrp18 in retaining intron 3 in ats1+ transcripts in vivo. Among the ten microarray probes, custom-designed to detect specifically the mRNA isoforms arising from altered use of donor 5’ splice sites, we were able to detect in wild-type cells the utilisation of a downstream alternate 5’ss in intron 1 of D-Tyr-tRNA deacylase. Comparative assessment of this splicing event in prp18-5int and slu7-2 mutant cells revealed that SpPrp18 is preferentially required for the utilisation of its alternative 5’ss and such a role has not yet been attributed to its budding yeast and human homologs. On the other hand, SpSlu7 was required equally for utilisation of canonical and non-canonical 5’ss. Differential requirement for SpSlu7 for the utilisation of an upstream non-canonical 3’ss and the canonical 3’ss in DUF3074 intron 1, was noted. This role of SpSlu7 in 3’ss selection is similar to that known from in vitro studies of its budding yeast and human counterparts. Overall, we identified and experimentally validated novel alternate splice events in fission yeast and we infer an important role for SpSlu7 and SpPrp18 in both 5’ss and 3’ss selection.

Over the past decade several converging lines of evidence have highlighted the importance of post-transcriptional events in skeletal muscle. This level of regulation is controlled by multi-functional RNA-binding proteins and trans-acting factors. In fact, several RNA-binding proteins are implicated in neuromuscular disorders including myotonic dystrophy type I, spinal muscular atrophy and amyotrophic lateral sclerosis. Therefore, it is necessary to examine the impact of RNA-binding proteins during skeletal muscle development and plasticity in order to understand the consequences linked to their misregulation in disease. Here, we focused on the RNA-binding protein Staufen1, which assumes multiple roles in both skeletal muscle and neurons. We previously demonstrated that Staufen1 is regulated during myogenic differentiation and that its expression is increased in denervated and in myotonic dystrophy type I skeletal muscles. The increased expression of Staufen1 initially appeared beneficial for DM1 since further elevating Staufen1 levels rescued key hallmarks of the disease. However, based on the multi-functional nature of Staufen1, we hypothesized that Staufen1 acts as a disease modifier in DM1. To test this, we investigated the roles of Staufen1 in skeletal muscle biology and their implications for disease. Our data demonstrated that Staufen1 is required during the early stages of muscle development, however its expression must remain low in postnatal skeletal muscle. Interestingly, the overexpression of Staufen1 impaired myogenesis through the regulation of c-myc translation. Since the function of c-myc in oncogenesis is well described, we investigated the role of Staufen1 in cancer biology. In particular, we determined novel functions of Staufen1 in rhabdomyosarcoma tumorigenesis, thus providing the first direct evidence for Staufen1’s involvement in cancer. Moreover, based on Staufen1’s role in myogenic differentiation and in myotonic dystrophy type I, we generated muscle-specific transgenic mice to examine the impact of sustained Staufen1 expression in postnatal skeletal muscle. Staufen1 transgenic mice developed a myopathy characterized by histological and functional abnormalities via atrogene induction and the regulation of PTEN mRNAs. In parallel, we further investigated Staufen1-regulated alternative splicing and our data demonstrated that Staufen1 regulates multiple alternative splicing events in normal and myotonic dystrophy type I skeletal muscles, both beneficial and detrimental for the pathology. Collectively, these findings uncover several novel functions of Staufen1 in skeletal muscle biology and highlight Staufen1’s role as a disease modifier in DM1.

Le genre des Deltaretrovirus est composé des virus de la leucose bovine (BLV) et des virus T-lymphotropes de primates (PTLV-1, -2, -3 et -4) qui regroupent les virus humains T-lymphotropes (HTLV-1, -2, -3 et -4) et les virus simiens apparentés (STLV-1, -2, -3 et -4). Seules les infections par les PTLV-1 et BLV sont associées à des pathologies : une lymphoprolifération maligne appelée ATLL chez l'homme et le singe ou une maladie neurologique appelée HAM/TSP chez l'homme infectés par les PTLV-1 et une lymphoproliferation maligne chez les bovins infectés par BLV. L'infection par HTLV-2 n'est associée qu'à une lymphocytose et le pouvoir pathogène d'HTLV-3 n'est pas caractérisé à ce jour. Les lentivirus, parmi lesquels on trouve les agents étiologiques du SIDA VIH-1 et VIH-2, et les Deltaretrovirus, sont des rétrovirus complexes. Ils codent donc, en plus des protéines structurales et enzymatiques, pour des protéines régulatrices ainsi que pour des protéines auxiliaires qui seront au centre des travaux présentés. Chez HTLV-1 et BLV, les protéines auxiliaires jouent un rôle primordial dans l'infectiosité in vivo. Or ces protéines n'avaient pas encore été décrites chez les PTLV-3. Leur caractérisation composait donc le premier objectif de ces travaux de thèse. Nous avons ainsi identifié les ARN messagers codant 3 nouvelles protéines putatives in vitro. Nous avons étudié les caractéristiques de ces protéines, notamment leur rôle dans le cycle des PTLV-3 in vitro et leur expression in vivo. Dans un second travail, nous avons essayé de comprendre si les différents domaines fonctionnels déjà identifiés dans la protéine Vpx, une protéine auxiliaire de VIH-2 influençaient sa capacité à interagir avec le facteur de restriction cellulaire SAMHD-1. Nous avons voulu déterminer le compartiment cellulaire dans lequel Vpx induisait la dégradation de SAMHD-1 et la cinétique de ce phénomène, qui permet à ce virus de se répliquer dans les lignées myéloïdes. Ces travaux apportent des éléments nouveaux dans la compréhension du rôle des protéines auxiliaires sur la régulation fine du cycle rétroviral et l'échappement au système immunitaire inné / Deltaretroviruses include bovine leukemia viruses (BLV) and primate Tlymphotropic viruses (PTLV-1, -2, -3 and -4) which are composed of human Tlymphotropic (HTLV-1, -2, -3 and -4) and of their simian counterparts (STLV-1, -2, -3 and -4). PTLV-1 and BLV are the only ones associated to pathologies: a lymphoproliferative disorder named ATLL in humans and non human primates and a neurological disorder named HAM/TSP in humans in the case of PTLV-1 and a Bmalignant lymphoproliferation in BLV infected cattle. HTLV-2 has not been associated with any disease so far and the pathogenic potential of HTLV-3 remains unknown. Lentiviruses, including HIV-1 and -2 the AIDS etiological agents, and Deltaretroviruses, are complex retroviruses. Therefore, in addition to structural and enzymatic proteins they encode regulatory proteins and also auxiliary proteins, the main subject of this work. HTLV-1 and BLV auxiliary proteins play key roles in viral infection in vivo. Whether the genome of PTLV-3 encodes such proteins was not determined yet. Therefore their characterization was the first goal of my PhD work. We identified in vitro messenger RNAs encoding 3 new putative proteins. Their impact on the PTLV-3 viral life cycle in vitro and their expression in vivo were then investigated. As a second part of this work, we examined the relationship existing between the Vpx HIV-2 auxiliary protein and its ability to interact with a restriction factor named SAMHD-1. Vpx induces SAMHD-1 degradation and the kinetic of such degradation allows the virus to replicate in myeloid cells. Altogether, these projects provide new insights into the understanding of the roles played by retroviral auxiliary proteins in connection with a tight regulation of viral life cycle and an escape from innate immunity

Le diagnostic des maladies génétiques est difficile à émettre. En effet, il est souvent difficile de déterminer comment une mutation va entrainer la pathologie. Le but de cette thèse est de développer des outils permettant de répondre à cette interrogation. Les mutations peuvent entrainer des anomalies à différents niveaux, différentes outils ont ainsi été développées en parallèle afin de pouvoir détecter différents types d'anomalies. Ces outils ont été développés en utilisant comme modèle une maladie génétique appartenant à la famille des myopathies, entrainant une dégénérescence des muscles des patients. Les travaux de cette thèse ont permis de confirmer le caractère délétère de certaines mutations en démontrant des anomalies dans un mécanisme appelé « épissage » qui permet la transmission de l'information contenue dans le génome. Les mutations en empêchant cette transmission vont ainsi être responsables de la maladie. / Diagnosis of genetic diseases is a difficult task. Indeed, it is often difficult to determine if mutations detected in patients will be responsible of the disease. The aim of this thesis is to develop tools allowing answering on this question. Mutations can have deleterious effects to several levels, thus different tools have been develop in parallel in order to detect different kind of abnormalities. These tools have been developed using as model a genetic disease belonging to the family of myopathy, leading to a degeneration of patients muscles. These thesis works have confirmed the deleterious effect on some mutations in a mechanism named "splicing" which allow transmission of the genome's information. Mutations preventing the transmission will thus be responsible of the disease.

Chez Arabidopsis thaliana, les gènes d’ARNr 5S sont transcrits par l’ARN polymérase III qui fait intervenir plusieurs facteurs de transcription dont TFIIIA qui reconnaît spécifiquement le promoteur interne de ces gènes et permet ainsi le recrutement de l’ensemble du complexe de transcription. Le gène TFIIIA est composé de sept exons dont le troisième, résultant de l’exonisation d’une molécule d’ARNr 5S, est épissé de façon alternative produisant ainsi deux transcrits. Le transcrit ES, qui ne contient pas cet exon, code pour la protéine TFIIIA pleine longueur et fonctionnelle. Le transcrit EI, dans lequel l’exon « 5S-like » est maintenu, est reconnu et dégradé par la voie NMD (Non-Mediated Decay). L’exon « 5S-like » a, en effet, la particularité de contenir un codon stop prémature qui en fait une cible de la voie NMD. Lors de l’étude de l’expression des transcrits ES et EI ainsi que celle de l’accumulation de la protéine TFIIIA au cours du développement, nous avons montré que le taux de la protéine TFIIIA fonctionnelle est soumis à plusieurs niveaux de régulation. En effet, la production de la protéine TFIIIA pleine longueur est le résultat de la synthèse du transcrit ES et de sa traduction mais également de l’efficacité du clivage protéolytique de la protéine. Lors de la maturation de la graine, l’accumulation croissante de protéine TFIIIA résulte d’une augmentation des quantités de transcrits ES couplée à une diminution du clivage protéolytique. Dans les premiers jours du développement, la protéine TFIIIA n’est détectée qu’après le 4ème jour, suite à la diminution du clivage. Sa présence est corrélée au remodelage de la chromatine de l’ADNr 5S. La combinaison de ces deux mécanismes permet ainsi la production de TFIIIA et de son produit de transcription l’ARNr 5S en fonction des besoins de la plante. / In Arabidopsis thaliana, 5S rRNA genes are transcribed by RNA polymerase III. TFIIIA, specifically required for transcription of these genes, binds to the internal control region of the 5S rRNA genes and allows the assembly of the full transcription complex pol III. The TFIIIA gene consists of seven exons, the third of which results in the exonisation of one 5S rRNA molecule. This exon “5S-like” is alternatively skipped or included to produce either of two transcript isoforms. The ES transcript encodes the fully functional TFIIIA protein. The EI transcript, which contains the exon “5S-like”, is a target of the NMD pathway (Non-Mediated Decay). Indeed, the exon “5S-like” contains a premature stop codon, which is recognized by this RNA decay pathway. During the study of the ES and EI transcripts expression and the TFIIIA protein accumulation throughout the plant development, we show that TFIIIA functional protein levels are under control of many regulation steps. Actually the production of the full-length TFIIIA protein results from production and translation of the ES transcript but also from the proteolytic cleavage efficiency of TFIIIA protein. During the seed maturation, the strong accumulation of TFIIIA results from an increase in ES amounts and a proteolytic cleavage decrease. After the fourth day post-germination, TFIIIA protein is detected because the proteolytic cleavage decreases. TFIIIA presence is correlated with 5S rDNA chromatin reorganization. The combination of these two mechanisms allows TFIIIA production and its transcription product 5S rRNA according to the plants needs.

Orientadores: Íscia Teresinha Lopes Cendes, Cristiane de Souza Rocha / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T01:10:50Z (GMT). No. of bitstreams: 1 BrumattiGoncalves_KatiaCristiane_M.pdf: 3380943 bytes, checksum: e87dbdc3a9db8349ec00b7148c98fd7b (MD5) Previous issue date: 2015 / Resumo: O campo da bioinformática associada à Next Generation Sequencing (NGS) ainda está em estado imaturo. A técnica de microarray tem sido muito utilizada nas últimas décadas em estudos de níveis de expressão de genes, porém essa técnica possui limitações. Sequenciamento de RNA (RNA-Seq) tem vantagens sobre as abordagens atuais, pois permite que o transcriptoma inteiro seja pesquisado com alto rendimento, fazendo com que RNA-Seq seja útil para estudar transcriptomas complexos, além disso, permite a análise de splicing alternativo. Muitas ferramentas têm sido desenvolvidas para abordar diferentes aspectos da análise de dados em RNA-Seq, e sua análise é um desafio constante. Nesse contexto, o objetivo deste estudo foi utilizar métodos de bioinformática para a análise de expressão gênica utilizando dados de RNA-Seq. Para isso, foram utilizados dados brutos obtidos em dois experimentos diferentes: a) utilizando animais normais, na qual a análise comparativa foi realizada da região do hipocampo (CA1, CA2 e CA3) e giro denteado, e b) utilizando animais tratados com pilocarpina e animais controle. Na análise dos dois experimentos, foram encontrados 3 genes (Nnat, Sv2b e Neurod6) em comum que tem diferença na expressão, ambos genes tem envolvimento no sistema nervoso central. Na análise de splicing alternativo, a ferramenta MISO (Mixture of Isoforms) comparado ao pipeline utilizado em Cuffdiff, gerou resultados melhores e mais detalhados, já que a ferramenta também realiza a quantificação dos transcritos, e com seus resultados foram descobertos 6 transcritos (Arpp21, Gria1, Gria2, Nrxn1, Dclk1 e Rtn1) em comum nas regiões do hipocampo, que tem alta expressão em giro denteado. Atualmente, existem diversos softwares em ascensão para análise diferencial, porém, o pipeline utilizado neste trabalho é ainda uma das principais ferramentas para análise de RNA-Seq, por usar algoritmos confiáveis e permitir flexibilização das análises quando necessário. Este estudo apresentou uma proposta de pipeline para a análise de expressão diferencial e identificação de splicing alternativo, para dados obtidos através de tecnologia de sequenciamento RNA-Seq. Foram identificados 5760 transcritos considerados significativamente expressos, e sugere que 6 transcritos sejam decorrentes de splicing alternativo / Abstract: The field of bioinformatics associated with Next Generation Sequencing (NGS) is still in an immature state. The microarray technique has been widely used in recent decades in studies of gene expression levels, but this technique has limitations. Sequencing RNA (RNA-Seq) has advantages over current approaches because it allows the whole transcriptome is researched with high yield, making RNA-Seq be useful for studying complex transcriptomes, moreover, allows the analysis of alternative splicing. Many tools have been developed to aproach different aspects of data analysis in RNA-Seq, and its analysis is a constant challenge. In this context, the objective of this study was to use bioinformatics methods for gene expression analysis using RNA-Seq data. For this, the raw data obtained in two different experiments were used: a) using normal animalsin which was made a comparative analysis of the hippocampus (CA1, CA2 and CA3) and dentate gyrus, and b) using pilocarpine treated animals and animals control. In the analysis of two experiments, were found three genes (NNAT, Sv2b and Neurod6) in common that there is a difference in the expression, both of genes is involved in the central nervous system. In alternative splicing analysis, MISO (Mixture of Isoforms) tool compared to the pipeline used in Cuffdiff, gave better and more detailed results, as the tool also performs the quantification of transcripts, and their results were found 6 transcripts (Arpp21, Gria1, Gria2, Nrxn1, Dclk1 and Rtn1) in common in the regions of the hippocampus, which has high expression in the dentate gyrus. Currently, there are various software on the rise for differential analysis, however, the pipeline used in this work is still one of the main tools for RNA-Seq analysis, by using reliable algorithms and allow flexibility of analyzes when necessary. This study showed a pipeline proposed for the analysis of differential expression, and alternative splicing of identification data obtained for RNA-Seq sequencing technology. 5760 transcripts considered significantly expressed were identified, and suggests that 6 transcripts are derived from alternative splicing / Mestrado / Fisiopatologia Médica / Mestra em Ciências

No description available.

Splicing is the process when introns gets removed and exons are spliced together. This is an important step to form a clean mRNA with no unnecessary sequences that could interrupt protein synthesis. There are different types of splicing and some of them need a complex called spliceosome. The spliceosome requires ATP, small nuclear RNAs and splicing factors. The spliceosome and the process splicing can be regulated by epigenetics, and one epigenetic mechanism is histone modification. There are four types of histone modifications; methylation, phosphorylation, ubiquitination and acetylation. They regulate splicing to different extents by altering the chromatin structure, affect the assembly of the spliceosome and regulate the attraction of splicing factors. This review will investigate if histone modifications affect splicing and to what extent. Suggestions for further research regarding the relationship between splicing and histone modifications will also be provided. The review is based on 30 articles and two books and the search was conducted between 30th of March 2020 and 13th of April 2020. Ubiquitination and phosphorylation have a minor effect on splicing meanwhile methylation and acetylation affect splicing in great extent.

L’analyse des défauts d’épissage est particulièrement complexe. Outre la diversité des transcrits présents à l’état physiologique, les variations nucléotidiques peuvent induire des modifications hétéroclites de l’épissage. Ces variations, appelées variants splicéogéniques, et leur impact au niveau de l’épissage, sont à même de modifier plus ou moins sévèrement le phénotype de l’individu.Au cours de ce travail de thèse, nous nous sommes intéressés à trois grands aspects de l’étude des défauts de l’épissage : (i) la prédiction de ces défauts d’épissage, (ii) l’analyse des données de RNA-seq et (iii) le rôle de l’épissage dans l’interprétation de la pathogénicité d’un variant pour la prédisposition aux cancers du sein et de l’ovaire (syndrome HBOC).Nous avons optimisé les recommandations en vigueur pour identifier les variants splicéogéniques au sein des séquences consensus des sites d’épissage. Ce travail a conduit à la publication d’un nouvel outil SPiCE (Splicing Prediction in Consensus Elements), développé sur 395 variants. SPiCE a le potentiel d’être une aide à la décision pour guider les généticiens vers ces variants splicéogéniques, grâce à une exactitude de 94.4 %. Puis, nous avons comparé les outils de prédiction des points de branchement. Pour cela, une collection sans précédente de 120 variants avec leurs études ARN a été établi dans la région des points de branchements. Nous avons ainsi révélé que ces outils de prédictions sont aptes à prioriser les variants pour des études ARN dans ces régions jusque-là peu étudiées. Pour étendre les prédictions des variants splicéogéniques au-delà d’un motif spécifique, nous avons construit l’outil SPiP (Splicing Prediction Pipeline). SPiP utilise un ensemble d’outils pour prédire un défaut d’épissage quel que soit la position du variant. Ainsi, SPiP peut ainsi s’adresser à la diversité des défauts d’épissage avec une exactitude de 80.21 %, sur une collection de 2 784 variants.Les données issues du RNA-seq sont complexes à analyser, car il existe peu d’outils pour annoter finement les épissages alternatifs. Aussi nous avons publié l’outil SpliceLauncher. Cet outil permet de déterminer une grande diversité de jonctions d’épissage, indépendamment des systèmes RNA-seq utilisés. Cet outil renvoie aussi les résultats sous formes graphiques pour faciliter leur interprétation.Puis nous avons évalué le rôle de l’épissage alternative dans l’interprétation à usage clinique d’un variant. Le gène PALB2, impliqué dans le syndrome HBOC, a été utilisé comme modèle d’étude. Nous avons ainsi démontré que l’épissage alternatif de PALB2 est apte à remettre en cause la pathogénicité de certains variants. La collecte de données fonctionnelles et cliniques sont donc nécessaires pour conclure sur leur pathogénicité.Nos travaux illustrent ainsi l’importance de la caractérisation et de l’interprétation des modifications de l’épissage pour répondre aux défis présents et futurs du diagnostic moléculaire en génétique. / Analysis of splicing defects is particularly complex. In addition to the diversity of physiological transcripts, nucleotidic variations can induce heterogeneous alteration of splicing. These variations, called spliceogenic variants, and their impact on splicing, can involve severe consequences on the individual phenotype.In this thesis work, we focused on three main aspects of the study of splicing defects: (i) the prediction of these splicing defects, (ii) the analysis of RNA-seq data and (iii) the role of splicing in interpreting the pathogenicity of a variant for the hereditary breast and ovarian cancers (HBOC syndrome).We optimized the current recommendations to identify spliceogenic variants within the consensus sequences of splicing sites. This work led to the publication of a new tool, SPiCE (Splicing Prediction in Consensus Elements), developed on 395 variants. SPiCE has the potential to be a decision support tool to guide geneticists towards these spliceogenic variants, with an accuracy of 94.4%. Then, we compared the tools dedicated to branch points prediction. For this purpose, an unprecedented collection of 120 variants with their RNA studies has been established in the branch point region. Thus, we revealed these prediction tools are able to prioritize variants for RNA studies in these hitherto poorly studied regions. To extend the predictions of spliceogenic variants beyond a specific motif, we built SPiP (Splicing Prediction Pipeline) tool. SPiP uses a set of tools to predict a splicing defect regardless of the variant position. Thus, SPiP can address the diversity of splicing defects with an accuracy of 80.21%, on a collection of 2,784 variants.The data from the RNA-seq are complex to analyze, as there are few tools to finely annotate alternative splices. Also we published SpliceLauncher tool. This tool allows to determine a wide variety of splicing junctions, independently of RNA-seq systems used. This tool also returns the results in graphical form to make interpretation user-friendly.Then we evaluated the role of alternative splicing in the clinical interpretation of a variant. The PALB2 gene, involved in HBOC syndrome, was used as a study model. Thus, we demonstrated that the alternative splicing of PALB2 is able of challenging the pathogenicity of certain variants. Collection of functional and clinical data is therefore necessary to conclude on their pathogenicity.Our work thus illustrates the importance of characterizing and interpreting splicing modifications to meet the current and future challenges of molecular diagnosis in human genetics.

L'expression des ARN mitochondriaux et chloroplastiques des plantes implique un grand nombre de modifications post-transcriptionnelles, parmi lesquelles l'épissage des introns est un processus essentiel. Sur la base de leur structure et des mécanismes d'épissage associés, les introns peuvent être classés en deux familles et ceux présents dans les organites des plantes appartiennent au groupe II. Les introns mitochondriaux et chloroplastiques de groupe II sont fortement dégénérés et ont perdu la capacité de s'auto-épisser in vivo. Leur élimination nécessite l’action de nombreux facteurs protéiques codés dans le noyau et importés dans les organites. Les protéines de liaison à l'ARN jouent un rôle prédominant dans ce processus complexe. Les protéines ribosomales sont des protéines abondantes se liant à l'ARN et peuvent être recrutées pour remplir diverses fonctions annexes. Au cours de ma thèse, j’ai étudié la fonction des protéines de type uL18 chez Arabidopsis, qui comprend 8 membres. Ces protéines partagent un domaine uL18 plutôt dégénéré, mais dont la structure est conservée, et dont la fonction initiale est de permettre l’association avec l’ARNr 5S. Nos résultats ont montré que cinq protéines de type uL18 sont adressées aux mitochondries et trois aux chloroplastes. Deux d’entre elles correspondent à de véritables protéines ribosomales uL18 associées aux ribosomes des organites, tandis que deux autres (uL18-L1 et uL18-L8) se sont transformées en facteurs d'épissage et sont nécessaires à l'élimination d’introns mitochondriaux ou chloroplastiques spécifiques. L'analyse d'un troisième membre de la famille, uL18-L5, a révélé qu'il participait à l'épissage de nombreux introns mitochondriaux. Mes résultats ont permis de révéler que les facteurs dérivés des protéines ribosomales uL18 jouent un rôle essentiel dans l’épissage des introns du groupe II mitochondriaux ou chloroplastiques chez les végétaux et que ces fonctions ciblent sot un seul intron ou bien plusieurs d’entre eux. / RNA expression in plant organelles implies a large number of post-transcriptional modifications in which intron splicing is an essential process. Based on RNA structures and splicing mechanisms, introns can be classified into two families and organellar introns of seed plants are categorized as group II. Organellar group II introns are highly degenerate and have lost the ability to self-splice in vivo. Their removal from transcripts is thus facilitated by numerous nuclear-encoded proteins that are post-translationaly imported into organelles. Among them, RNA binding proteins play predominant roles in this complex process. Ribosomal proteins are abundant RNA-binding proteins and could be recruited to carry out multifarious auxiliary functions. During my thesis, I investigated the function of the uL18 ribosomal-like protein family in Arabidopsis that comprises 8 members. The members of this protein family share a rather degenerate but structurally conserved uL18 domain whose original function is to permit association with the 5S rRNA. Our results showed that five uL18-Like proteins are targeted to mitochondria and three to chloroplasts. Two of these proteins correspond to real ribosomal uL18 proteins that incorporate into organellar ribosomes, while two other members (uL18-L1 and uL18-L8) have turned into splicing factors and are required for the removal of specific mitochondrial or plastid group II introns. The analysis of a third member, uL18-L5, revealed that it participated in the splicing of numerous mitochondrial introns. Our results revealed that uL18-like factors play essential roles in group II intron splicing in both mitochondria and plastids of plants and that these functions could target a single or multiple introns.