Pre-mRNA splicing is a highly regulated cellular process. The tight cooperation of spliceosome and other splicing factors that enable pre-mRNA cis-elements interpretation results in precise pre-mRNA splicing regulation. Short conserved splicing sequences within introns represent an elementary and indispensable element for intron removal from primary transcript, yet they are not sufficient signals for efficient splicing events. Additional pre-mRNA features affect complex splicing regulation. We took advantage of strains with slightly disrupted spliceosome (prp45(1-169)) to study the effect of ACT1 and MAF1 intronic sequences on splicing efficiency. Here we show, that ACT1 intron region between branch point (BP) and 3' splice site (3'ss) maintains splicing efficiency in mutant cells. However, the specific element within this region was not determined. In addition, results implicate an alternative BP in splicing efficiency modulation in yeast Saccharomyces cerevisiae. Interestingly, this alternative BP is localized in ACT1 intron outside of the BP-3'ss region. Furthermore, splicing factors with potential influence on 3'ss selection were studied. Heterodimer composed of Slu7p and Prp18p participates in 3'ss positioning to the active site of the spliceosome. Splicing analysis of substrates with two...

Depuis plusieurs décennies, l’étude des conséquences des mutations pathogéniques a permisnon seulement de définir l’origine de nombreuses maladies génétiques humaines, héréditaires ou non,mais également de contribuer à l’interprétation de la variabilité phénotypique inter-individus au seind’une pathologie donnée. Les mutations induisant une perte de fonction de la protéine synthétisée ouune protéine incomplète ont été et sont encore les plus étudiées. Avec l’introduction des technologiesde séquençage à haut-débit, le nombre de variants faux-sens, silencieux ou introniques détectés auniveau des gènes humains augmente de façon continue. Distinguer les variations nucléotidiques quivont modifier significativement un phénotype de celles qui seront neutres est un véritable défi pour larecherche.De nombreuses variations nucléotidiques à signification clinique inconnue ont été identifiéesparmi les près de 2000 mutations décrites au niveau du gène CFTR (Cystic Fibrosis Transmembraneconductance Regulator) responsables de la mucoviscidose. Certaines de ces variations vont avoir unimpact sur les transcrits ARN modifiant leur qualité et leur quantité et par conséquent l’expression dugène CFTR. Elles vont notamment affecter l’épissage de l’ARN pré-messager en altérant des signauxreconnus par la machinerie cellulaire et perturber la fidélité de ce mécanisme. Malgré un recul de plusde 20 ans dans la description de la corrélation génotype-phénotype dans cette maladie, de nombreuxphénotypes inattendus et atypiques sont observés. Ils peuvent être dus à des facteurs autres que desvariations génotypiques, mais sans l’accès direct aux transcrits des individus porteurs de tels variants,il est difficile de mesurer les conséquences de ces variations sur la synthèse et la maturation de l’ARN.L’objectif de ce travail de thèse a été de montrer, par des approches in vitro etbioinformatiques, comment des variations nucléotidiques au sein du gène CFTR peuvent impacter surl’épissage de l’ARN pré-messager. Ce travail a permis dans un premier temps la découverte demécanismes d’épissage inattendus modificateurs de phénotypes sévères attendus pour une mutationnon-sens. En effet, par un épissage alternatif subtil de trois nucléotides au niveau d’un site d’épissageen tandem, le codon stop se retrouve délété et aboutit à des transcrits fonctionnels expliquant lesphénotypes modérés. Dans un second temps, nous avons montré que plusieurs mutations non-sensportées par le même exon 15 impactaient différemment sur l’épissage de l’ARN en modifiantsignificativement le taux d’inclusion de cet exon. La connaissance préalable des ratios de transcritsincluant ou non l’exon peut améliorer l’efficacité des traitements correcteurs de codons stopprématurés en les combinant avec des traitements modulateurs de l’épissage augmentant le taux detranscrits correctibles. Dans un troisième et dernier temps, une combinaison d’analyses in silico et invitro des exons du gène CFTR a permis de détecter des exons porteurs de signaux d’épissage plusfaibles les rendant plus sensibles à des mutations exoniques d’épissage. Des mutations faux-sens auniveau de l’exon 3 ont par exemple été montrées comme favorisant l’exclusion de cet exon, diminuantle taux de transcrits fonctionnels.L’ensemble de ces travaux a contribué à comprendre les conséquences de mutations au niveaude l’épissage du gène CFTR sur les variations du phénotype. La connaissance améliorée des variationspossibles du phénotype rattaché à un génotype donné permettra non seulement de prédire l’évolutionde la maladie, mais également d’ajuster et de proposer des thérapies personnalisées selon la mutationportée par le patient. / Over the past decades, studying the consequences of pathogenic mutations has allowed not only todefine the origin of several genetic diseases, but also to contribute to understand the phenotypicvariability between individuals within a disease. The CFTR gene was extensively analyzed since 1989,but among the over 1,900 mutations identified, the current challenge is to classify them as diseasecausingor neutral. These variants of unknown clinical significance (UVs) can alter multiple processes,from gene transcription to RNA splicing or protein function. The CFTR gene needs to include intactversions of all its 27 exons to be functional, and any mutation affecting its splicing process will reducethe amount of functional full-length transcripts. Previous studies have shown that mutations withinsome CFTR exons increase exon skipping. We hypothesized that a number of UVs occurring in otherCFTR exons could indeed affect splicing. By combining in vitro and bioinformatics approaches, weshowed how nucleotide variations within the CFTR gene could impact on the splicing of its premRNA.This work enabled us to provide the first experimental evidence of a premature terminationcodon removal by alternative splicing at a NAGNAG acceptor splice site. This unexpected phenotypemodifyingmechanism explains the much milder phenotype severity than expected for a nonsensemutation. The correction of premature termination codons (PTCs) by agents that promote readthroughrepresents a promising emerging tool for the treatment of many genetic diseases. Having demonstratedthat nonsense mutation could cause aberrant splicing, we postulated that the efficiency of thereadthrough treatment could be due not only to the stop codon itself but also to the amount ofcorrectible transcripts. We showed that a subset of nonsense mutations within the CFTR exon 15 has adifferent impact on the splicing efficiency by modifying the inclusion rate of this exon anddemonstrated that the total amount of transcripts together with the splicing profile should be assessedto anticipate and improve efficacy of readthrough therapy in CF patients. Finally, in order to anticipatethe occurrence of “splicing-causing” mutations, we used a combination of in silico and in vitroanalyses of all CFTR exons and pinpointed those harboring weak splicing signals, which render themmore sensitive to exonic splicing mutations.All these studies contribute to expand our knowledge on the phenotypic variability due to alternativesplicing of the CFTR gene. These studies will lead not only to predict the evolution of a disease, butalso to adjust therapy according to the mutation of each patient.

Pre-mRNA splicing is a vital process of gene expression important for all eukaryotic organisms. For the proper function of this very complex and dynamic event the presence of few specialized RNA and many proteins that hold a variety of tasks is necessary, not only inside the splicing complex itself, but also beyond this complex. The Prp45 is one of the proteins involved in pre-mRNA splicing in yeast Saccharomyces cerevisiae. Its human homologue, SNW1/SKIP, is involved in splicing but also in other crucial cell processes. The Prp45 protein was reliably reported only to participate in the second transesterification reaction of splicing. But there are also data suggesting its possible involvement in the first transesterification reaction. This work provides further evidences linking protein Prp45 with the first splicing reaction, obtained by the research of cells carrying the mutant allele prp45(1-169). Cells carrying this allele show dropped splicing and accumulation of pre-mRNAs. This thesis therefore also investigated the possible influence of Prp45 protein on the RNA export from the nucleus to the cytoplasm. But no connection between this protein and RNA transport was discovered. Keywords pre-mRNA splicing; Saccharomyces cerevisiae; Prp45; Mer1; Mud2; Prp22; Rrp6; AMA1; SNW1/SKIP

The Breast cancer gene 1 (BRCA1) codes for nuclear phosphoprotein with a key function in the regulation of DNA damage response. The BRCA1 protein contributes to the formation and regulation of protein supercomplexes that participates on the DNA double-strand break repair. These protein supercomplexes are formed by the protein-protein interactions between highly conservative protein motives in BRCA1 and its binding partners. Except to the wild type form of BRCA1 mRNA containing entire set of 22 exons coding for the 220 kD protein, numerous alternative splicing variants (ASVs) BRCA1 mRNA has been described. These ASVs code for BRCA1 isoforms lacking several critical functional domains. It has been proposed, that formation of BRCA1's ASVs represent a tool for regulation of BRCA1 function. Only poorly has been characterized a complex catalogue of in various human tissues and their expression. This study aims to address these questions. We optimized the identification of BRCA1's ASVs including those covering the entire transcripts of the wt BRCA1 mRNA with length exceeding 5.5 kb. In further analysis, we characterized 13 BRCA1's ASVs in RNA samples isolated from peripheral blood mononuclear cells (PBMNC) obtained from patients with breast cancer (BC) and control subjects. The majority of the identified...

hPrp8 is an essential pre-mRNA splicing factor. This highly conserved protein is a component of the U5 small ribonucleoprotein particle (U5 snRNP), which constitutes one of the building blocks of the spliceosome. hPrp8 acts as a key regulator of spliceosome activation and interacts directly with U5 snRNA and with the regions of pre-mRNA that are involved in the transesterification reactions during splicing. Mutations in hPrp8 have been shown to cause an autosomal dominant form of retinitis pigmentosa (RP), an inherited disease leading to progressive degeneration of retina. In this study, we analyzed the effects of the RP-associated mutations on the function of hPrp8. Using BAC recombineering, we created mutant variants of hPrp8-GFP construct and we generated stable cell lines expressing the recombinant proteins. The mutant proteins were expressed and localized to the nucleus. However, one of the missense mutations affected the localization and stability of hPrp8. Further experiments suggested that RP-associated mutations affect the ability of hPrp8 to interact with other components of the U5 snRNP and with pre-mRNA. We further studied the biogenesis of U5 snRNP. We depleted hPrp8 by siRNA to interfere with U5 snRNP assembly and we observed that the incompletely assembled U5 snRNPs accumulate in...

L'épissage alternatif des ARNs pré-messagers est le mécanisme majeur de diversification de l'information génétique chez les eucaryotes supérieurs. Près de 70% des gènes sont concernés par ce mécanisme. Il arrive malgré tout que l'épissage alternatif soit dérégulé ce qui conduit à des défauts d'épissage qui provoquent des maladies telles que le syndrome progérique de Hutchinson-Gilford (HGPS). Cette maladie se caractérise notamment par une apparition précoce des signes distinctifs de la vieillesse ainsi que de défauts métaboliques majeurs. Mon travail de thèse a consisté à sélectionner et à caractériser des composés chimiques modulateurs de l'épissage alternatif en se basant sur le mécanisme moléculaire conduisant à la progeria. Par la suite, l'efficacité de ces molécules a été testée dans des modèles de souris obèses. Parmi une chimiothèque de plus de 700 molécules chimiques, j'ai pu sélectionner sur différents critères une vingtaine de molécules modulatrices de l'épissage de l'exon 11 du gène LMNA. Une seule molécule a été capable de diminuer la prise de poids de souris mises sous régime hyperlipidique en favorisant la mise à disposition des lipides et en diminuant la différenciation adipocytaire grâce à son action sur les micros ARN. / Alternative splicing of pre-messenger RNA is the major mechanism to increase genetic variability in superior eukaryotes. Almost 70 % of genes are concerned by the mechanism. In some case this tightly regulated process is deregulated leading to splicing defects and diseases like Hutchinson-Gilford Progeroid Syndrome (HGPS). HGPS is characterized by a premature ageing and important metabolism defects.My thesis work was to select and characterized chemical compounds modulating alternative splicing based on molecular mechanism leading to HGPS. We further tested efficiency of these molecules in diet-induced obesity mice.Among a library of 700 compounds, I was able to select twenty molecules modulating splicing event of LMNA gene exon 11. One unique molecule was found to decrease weight gain in a mouse model under high-fat diet condition trough increasing lipolysis and lipids mobilization, and decreasing adipocyte differentiation.

Chez l'homme, l'épissage alternatif (EA) affecte presque tous les gènes permettant de générer de vastes répertoires d'ARN et de protéines. L'épissage est un processus hautement régulé qui s'effectue principalement lorsque l'ARN est en cours de synthèse sur la chromatine. Beaucoup d'études suggèrent que la chromatine et ses marques épigénétiques influencent les décisions d'épissage au locus correspondant. A l'inverse, d'autres données laissent penser que l'épissage peut moduler les marques épigénétiques. Au cours de ma thèse, j'ai étudié différentes voies de couplage entre l'épissage et la chromatine. D'une part, j'ai exploré l'impact de la méthylation de l'ADN sur la régulation de l'épissage. J'ai montré que les enzymes qui méthylent l'ADN ont un effet global sur l'épissage d'exons enrichis en méthylation. Mes données suggèrent que les protéines qui lient la méthylation de l'ADN sont impliquées dans cette régulation. D'autre part, j'ai exploré les conséquences de l'EA sur la régulation de la chromatine en étudiant son impact de deux histones-methyltransferases (HMTase) : G9A et SUV39H2 dont les gènes génèrent des transcrits alternatifs. Tous les transcrits variants codent pour des protéines. La conservation des variants d'épissage de G9A dans des espèces et l'absence de différences dans leur activité HMTase, nous amènent à proposer que l'EA est associé à une fonction non liée aux histones. A l'inverse, les isoformes de SUV39H2 exhibent des activités HMTases différentes et régulent l'expression de gènes cibles différents. Ensemble, nos résultats apportent de nouvelles connexions dans le couplage épissage-chromatine et supporte un modèle où ces derniers s'auto-influencent. / In humans, alternative splicing affects almost all genes in the genome and generates extensive repertoires of RNAs and proteins. Splicing is a highly regulated process which occurs primarily when the RNA is being synthesized on chromatin. Many studies suggest that chromatin and epigenetic marks influence splicing choices to the corresponding locus. Conversely, other data suggest that splicing can modulate epigenetic marks. During my thesis, I studied different ways of crosstalk between splicing and chromatin. First, I investigated the effect of DNA methylation on splicing regulation. I have shown that the enzymes that methylate DNA have an overall effect on the splicing of exons with enriched methylation. My data suggest that proteins which bind to methylated DNA are involved in this regulation. On the other hand, I explored the impact of alternative splicing on chromatin regulation studying its impact on the expression and activity of both histone methyltransferases (HMTase): SUV39H2 and G9A. G9A and SUV39H2 generate variants transcripts whose expression is regulated according to tissues. All variants transcripts encode proteins. Conservation of G9A splice variants in species and no differences in their HMTase activity, lead us to propose that G9A alternative splicing is associated with a non-histone function. Conversely, SUV39H2 isoforms exhibit different HMTases activities, and regulate the expression of different target genes. All our results provide new connections in chromatin - splicing coupling and support a model in which they harbor self-influence.

L’épissage, une étape majeure de l’expression des gènes, consiste en l’élimination des introns et la production de transcrits matures ou ARNm. La régulation ou des perturbations de l’épissage sont impliquées dans de nombreuses situations physiopathologiques. Dans ce travail, j’ai utilisé et analysé par des approches de bio-informatiques un grand nombre de données générées à large échelle afin de mieux définir les règles gouvernant la reconnaissance des exons au cours de l’épissage. Je montre que les mécanismes de reconnaissance des exons dépendent du biais de la composition nucléotidique des gènes qui les hébergent. Ainsi, la reconnaissance des exons hébergés par des gènes enrichis en guanine et cytosine dépend essentiellement de leur site 5’ d’épissage qui peut être masqué par des structures secondaires. La reconnaissance des exons hébergés par des gènes enrichis en thymine et adénine dépend essentiellement des signaux d’épissage situés en amont des exons. Je montre également que l’organisation chromatinienne est différente selon les biais de composition nucléotidique des gènes et que cela a un impact spécifique sur la reconnaissance des exons. De nombreuses études démontrent que les gènes ne sont pas organisés de façon aléatoire dans un génome et que l’architecture des gènes et des chromosomes dépend de leur composition nucléotidique. Par conséquent, mes travaux suggèrent qu’il existe un lien direct entre composition nucléotidique d’une région du génome, architecture de la chromatine et sélection des exons au cours de l’épissage. / Splicing, a major step in gene expression, consists in the removal of the introns and the production of mature transcripts or mRNA. The regulation of or the disturbances in splicing are involved in numerous physiopathological situations. In this work, I used and analysed with bio-informatic approaches a lot of genome-wide datasets in order to define better the rules governing exon recognition during the splicing step. I show in this work that the mechanisms of exon recognition depend on the nucleotidic composition bias of the genes which host these exons. Thus, the recognition of the exons located in genes enriched with guanine and cytosine essentially depends on their 5' splicing site, which can be hidden by secondary structures. The recognition of the exons located in genes enriched with thymine and adenine essentially depends on splicing signals placed upstream the exons. Moreover, I show that the chromatin organization varies according to the nucleotidic composition bias in the genes, and that it has a particular impact on exon recognition. A lot of studies have shown that the genes are not randomly organized in a genome and that the architecture of the genes and of the chromosomes depends on their nucleotidic composition. Put together, my work suggests that it exists an direct link between the nucleotidic composition of a genomic region, the chromatin architecture and the recognition of the exons during the splicing step.

In the process of pre-mRNA splicing introns are removed from pre-mRNA and exons are joined together. Current studies show, that about 95 % of genes, which contain more than two exons, can undergo alternative splicing. In this process some exons are included in or excluded from the final mRNA. Majority of pre-mRNA splicing take place co- transcriptionaly at this time RNA polymerase II is still attached to pre-mRNA. Alternative splicing is complex process that takes place in a close proximity of DNA and histones that might modulate alternative splicing decisions. Futher studies have validated fibronectin gene (FN1) and his alternative exons EDA and EDB (extra domain A and B) as suitably model for studying alternative splicing. Study using FN1 minigene reporter system, which is composed from EDA exon and two surrounding introns and exons, has proved that insertion of transcription enhancer SV40 infront of promotor, the level of EDA inclusion is decreased. So far, has not been prooved if this mechanism can function in real genome context and if distal transcription elements can influence alternative splicing. In this study, we have predicted transcription enhancer for FN1 gene by using The Ensemble Regulatory Build and FANTOM 5. The predicted transcription enhancer, is located 23,5 kbp upstream of TSS...

Ácido Hialurônico (HA) é um componente da matriz extracelular, responsável pela hidratação e manutenção do equilíbrio osmótico tecidual. Concentrações de HA estão elevadas em vários tipos de cânceres, incluindo próstata. Hialuronidases (HAases), são uma família de enzimas relacionadas com a propagação de infecções bacterianas, toxinas de venenos e progressão tumoral. A quebra do HA em pequenos fragmentos (3-25 dissacarídeos) promovidos pela ação das HAases tipo Hyal1, Hyal2 e Hyal3, está relacionada à promoção do câncer através da indução da angiogênese e estímulo a proliferação através de ativação da via tirosina quinase. Algumas isoformas de HAases, descritas como produto de splicing alternativo, possuem atividade enzimática diversificada. A heterogeneidade de expressão das HAases foi identificada em alguns tipos de câncer e pode ser correlacionada com o comportamento diferenciado dos tumores. Para este trabalho estudamos amostras de 55 pacientes submetidos a prostatectomia radical por carcinoma de próstata (CP) . A média de idade foi 66 anos e o tempo médio de seguimento 73,7 meses. Os pacientes foram divididos em dois grupos para análise dos resultados: 1- Escore de Gleason (EG) >=7 (30) e EG <=6 (25). 2- Comportamento tumoral (recidiva-19, e não recidiva-36), considerando o nível sérico de Antígeno Específico da Próstata (PSA) 0,2 ng/mL. O grupo controle foi representado por 11 pacientes com hiperplasia prostática benigna, submetidos à ressecção retropúbica. As HYAL foram identificadas por PCR, com uso de primers específicos para as variantes 1, 2, 3, 4 e 5 e wt da HYAL1, wt da HYAL2, e wt e variantes 1, 2 e 3 da HYAL3. As HYAL mais freqüentemente expressas pelo CP foram HYAL2-wt (65,4%), HYAL1-v1 (63,3%) e HYAL3-wt (47,2%). Em tecidos prostáticos benignos, a HYAL3-v1 foi expressa em 90,9% dos casos, estando presente em 36% dos tumores com EG baixo, e não expressa em tumores com EG alto (p=<0,001). Nos tumores sem recidiva HYAL1-v3 foi expressa em 30,5% dos casos versus 5,2% em casos que recorreram (p=0,041). HYAL3 v2, foi expressa por 33,3% dos tumores que não recorreram e não expressa em tumores que recorreram (p=0,002). Concluímos que a expressão de HYAL1-v3, HYAL3-v1 e HYAL3-v2 está relacionada a tumores mais diferenciados e com menores taxas de recidiva, podendo ser utilizadas como marcadores na prática clínica identificando candidatos a terapias mais conservadoras. / Hyaluronic acid (HA) is a component of the extracellular matrix that hydrates and maintains the osmotic balance of tissues. HA concentration is elevated in several cancers including prostate. Hyaluronidases (HAases) are a family of enzymes related to the spread of bacterial infections, toxins of venoms and probably cancer progression. Small fragments of HA are generated by HAase Hyal1, Hyal2 and Hyal3, stimulating endothelial proliferation and activating mitogen-activated protein kinase pathway. Several isoforms of HAases have been described as a product of alternative splicing, and are responsible for differences in the enzyme activity. The heterogeneity of HAses expression has been identified in tumors and could be related to the differences in their biological behavior. Fifty-five patients submitted to radical prostatectomy for prostate cancer (PC) were the subject of this study. The mean age was 66 years old and the mean follow-up was 73,7 months. Patients were divided into two groups for the analyses: 1- High Gleason score (GE) >=7 (30) and low Gleason score <=6 (25). 2- Tumor behavior; recurrence - 19 and nonrecurrence - 36. Biochemical recurrence was considered when PSA was higher than 0.2 ng/mL. The control was represented by 11 patients submitted to retropubic prostate resection for benign prostatic hyperplasia. The alternative splicing forms of HYAL were identified by PCR, and the primer sequences identified variants 1, 2, 3, 4, 5 e wt of HYAL1, wt of HYAL2, wt and variants 1, 2 and 3 of HYAL3. The HYAL more frequently expressed by PC was HYAL2-wt (65.4%), HYAL1-v1 (63.3%) and HYAL3-wt (47.2%). In benign prostate tissue the main expressed HAase was HYAL3-v1 in 90.9%, being present in 36% of low Gleason score tumors and not expressed by tumors with high Gleason score (p=<0.001). For tumors that not recurred there was expression of HYAL1-v3 in 30.5% of the cases vs. 5.2% in cases that recurred (p=0.041). The same difference was noted regarding the expression of HYAL3-v2, that was expressed by 33.3% of tumors that not recurred and not expressed by tumors that recurred (p=0.002). We conclude that there is a profile of HAase related to low Gleason score and non-recurrent PC that is characterized by expression of HYAL1-v3, HYAL3-v1 and HYAL3-v2 that could be used in clinical practice to choose a better treatment.