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La plupart des gènes humains codants génèrent des transcrits alternatifs (isoformes) par épissage alternatif (alternative splicing, AS) et polyadénylation alternative (APA) en général dans la région codante et la région 3’ non traduite (3’UTR), respectivement. Le rôle de l’AS et la 3’UTR-APA est de plus en plus reconnu dans l’oncogenèse. En particulier, des réseaux d’AS connectant des facteurs d’épissage et des variants d’épissage ont récemment été identifiés. L’AS est aussi largement régulé par les agents anticancéreux génotoxiques, tel que la doxorubicine et le cisplatine (induisant des différents types de lésions sur l’ADN), qui sont régulièrementt utilisés dans les traitements du cancer du sein et du poumon non-à-petites-cellules (non-small-cell lung cancer, NSCLC), respectivement. Étant donné l’apparition fréquente de résistances aux chimiothérapies, comprendre les mécanismes sous-jacents est crucial pour surmonter ce problème clinique. Il existe des exemples d’évènements d’AS associés à la résistance aux agents anticancéreux, mais l’implication des facteurs d’épissage et des réseaux d’AS est très peu connue. De plus, une étude précédente a démontré que la doxorubicine réprime un grand groupe d’exon terminaux alternatifs (alternative last exons, ALE), qui correspondent à l’utilisation de sites de polyadénylation introniques (intronic polyadenylation, IPA). Les ALEs ont un rôle émergent dans le cancer, mais on ne sait encore que très peu sur leur régulation par d’autres agents anticancéreux, tel que le cisplatine. Afin de mieux comprendre le rôle des régulations d’AS et d’APA dans la réponse et la résistance cellulaire à la chimiothérapie, mon projet de thèse avait deux objectifs principaux : 1) déterminer l’étendue, les réseaux régulateurs, et les fonctions des régulations d’AS dans la résistance à la doxorubicine des cellules de cancer du sein, et 2) déterminer l’étendue, les mécanismes, et l’impact des régulations d’ALE en réponse au cisplatine dans des cellules de NSCLC. Dans la première partie, j’ai identifié par RNA-seq des milliers d’évènements d’AS et des dizaines de facteurs d’épissage régulés dans un modèle cellulaire de cancer du sein ER+ résistant à la doxorubicine. Par un miniscreen siARN, j’ai identifié deux facteurs, ZRANB2 et SYF2, impliqués dans la résistance à la doxorubicine. D’autres analyses RNA-seq ont révélé les évènements d’AS régulés par ces deux facteurs peu étudiés, ainsi que leur convergence vers l’exon 5 alternatif de l’oncogène ECT2. La déplétion de ZRANB2, SYF2, et du variant ECT2-ex5 réduit l’arrêt en phase S induit par la doxorubicine et la résistance des cellules. De plus, un niveau élevé d’inclusion de l’exon 5 d’ECT2 corrèle avec une mauvaise survie spécifiquement de patientes ER+ traitées par chimiothérapie. Dans la deuxième partie, j’ai identifié par 3’-seq que le traitement cisplatine (mais pas oxaliplatine) induit des ALEs/IPAs dans des milliers de gènes enrichis en gènes de cycle et de mort cellulaire. Cet effet est lié à une inhibition de la processivité de l’élongation dans les longs gènes. Une analyse 3’-seq sur polysomes m’a permis de montrer que ces régulations d’ALEs impactent le traductome, et a révélé un groupe d’isoformes particulièrement courtes peu efficacement traduites, dont un transcrit connu avec une fonction non-codante. En conclusion, j’ai pu identifier durant ma thèse un nouveau réseau d’AS impliqué dans la résistance à la doxorubicine des cancers du sein ER+, et une importante régulation d’ALEs impactant le traductome en réponse au cisplatine dans des cellules NSCLC. Ces travaux améliorent notre compréhension du rôle de l’AS et des ALE/IPA dans la réponse et la résistance cellulaire à la chimiothérapie anticancéreuse. Au plus long terme, les transcrits alternatifs et les régulateurs identifiés constituent des biomarqueurs candidats de chimiorésistance. / Most human coding genes generate alternative transcripts (isoforms) through alternative splicing (AS) and alternative polyadenylation (APA), most often within the coding region and the 3’ untranslated region (3’UTR), respectively. Both AS and 3’UTR-APA regulations have been increasingly involved in oncogenesis. In particular, AS networks connecting oncogenic splicing factors and oncogenic splicing variants have been recently identified. AS is also widely regulated by genotoxic anticancer drugs, like doxorubicin and cisplatin that induce different types of DNA lesions and are widely used in breast cancer and non-small-cell lung cancer (NSCLC) therapy, respectively. Given the frequent occurrence of resistance to chemotherapy, understanding the underlying mechanisms is crucial to overcome this major issue. There are examples of AS events associated with anticancer drug resistance, but very little is known about the splicing factors and therefore the AS networks involved. In addition, a previous study showed that doxorubicin represses a large set of alternative last exons (ALE) corresponding to the use of intronic polyadenylation (IPA) sites. ALEs have an emerging role in cancer, but little is known about its regulation by other anticancer drugs, like cisplatin. In order to better understand the role of AS and APA regulation in cell response and resistance to chemotherapy, my PhD project had two main aims: 1) determine the extent, regulatory networks and function of AS regulation in breast cancer cell resistance to doxorubicin, and 2) determine the extent, mechanism and impact of ALE regulation in response to cisplatin in NSCLC cells. In the first part, I identified by RNA-seq thousands of AS events and dozens of splicing factors regulated in a cell model of acquired resistance to doxorubicin in ER+ breast cancer. Through an siRNA miniscreen, I found two splicing factors, ZRANB2 and SYF2, involved in doxorubicin resistance. Further RNA-seq analyses revealed the AS events regulated by depletion of these poorly characterized splicing factors, and their convergence on the alternative exon 5 of the oncogene ECT2. Depletion of ZRANB2, SYF2 and the ECT2-Ex5 variant reduces doxorubicin-induced S phase arrest and doxorubicin resistance. In addition, high inclusion levels of ECT2-Ex5 correlate with poor survival specifically in ER+ breast cancer treated with chemotherapy. In the second part, I found by 3’-seq that in NSCLC cell treatment with cisplatin (but not oxaliplatin) induces ALE/IPA in thousands of genes enriched in cell cycle and cell death. This effect is linked to an inhibition of transcription elongation processivity in long genes. 3’-seq analysis on polysomes showed that this ALE regulation impacts the translatome, and revealed a set of particularly short isoforms that were inefficiently translated, including a transcript with a non-coding function. In conclusion, during my thesis, I could identify a novel AS network involved in doxorubicin resistance in ER+ breast cancer, and widespread ALE regulation impacting the translatome in lung cancer cisplatin response. This work increases our understanding of AS and IPA role in cell response and resistance to anti-cancer chemotherapy. In the longer term, the identified alternative transcripts and regulators constitute candidate biomarkers of chemoresistance.

Le cancer colorectal (CRC) est l'un des cancers les plus agressifs. Afin de rechercher de nouveaux marqueurs de progression de ce cancer, des puces tout transcriptome et épissage, ainsi que des puces microARNs, ont été précédemment utilisées au laboratoire.Dans une première partie, l’analyse des données transcriptomiques a permis de trouver un ensemble de transcrits dérégulés dans les adénomes et dans le CRC, dont la surexpression de TIMP1, associée à la diminution de rétention de l’intron 3, par rapport à la muqueuse normale. Ce transcrit aberrant n’est pas sujet au mécanisme de dégradation active des ARNm («Nonsense-Mediated mRNA Decay »). Nous avons alors analysé les mécanismes d’épissage de TIMP1, ce qui nous a conduits à identifier hnRNPA1 comme un régulateur important, à la fois in vitro et in vivo, de la rétention de l’intron 3 de TIMP1, via sa fixation au début de l’exon 4. Le rôle de TIMP1i3 (+) dans la progression du CRC reste à identifier.Dans une seconde partie, l’analyse des données de la puce microARNs (miRs) a permis de trouver un ensemble de miRs dérégulés dans les CRC. Nous avons sélectionné des gènes codant des facteurs d’épissage dont l’expression était modifiée au cours de la progression cancéreuse, en tant que cibles possibles de certains de ces miRs. Six interactions ont été montrées (PRMT5/miR145; SRSF6/miR375; RBMX/miR23a; RBMX/miR24; RBMX/miR125a5p; SRSF11/miR143).En utilisant la technique de « Luciferase Reporter Gene Assay » couplée à la mutagenèse dirigée, nous avons montré que le miR145 régule négativement l’expression de PRMT5 par interaction avec la région 3’ non traduite du gène. Du fait du contrôle de la machinerie d’épissage des ARN prémessagers par PRMT5, nous proposons que miR145 pourrait être un régulateur général de l’épissage. / Colorectal cancer (CRC) is one of the most vaggressive cancers in the world. In order to look for new markers in progression of this cancer, all transcriptome and splice chips, as well as microRNA chips, have been previously used in the laboratory.In the first part of my thesis, transcriptomic data analysis revealed a set of deregulated transcripts in adenoma and in CRC, including overexpression of TIMP1, associated with the decrease of intron 3 retention, compared to normal mucosae. This aberrant transcript is not subject to the active mechanism of mRNAs degradation ("Nonsense-Mediated mRNA Decay"). Then we analysed the splicing mechanisms of TIMP1, which led us to identify hnRNPA1 as a major regulator, both in vitro and in vivo, of TIMP1 intron 3 retention via its binding in the beginning of exon 4. TIMP1i3 (+) role in the progression of CRC remains to be identified.In a second part, the analysis of microRNA data (miRs) revealed a set of deregulated miRs in CRC. The aim was to identify target genes for these miRs.We selected genes encoding splice factors whose expression was modified during cancer progression as possible targets for some of these miRs. Six interactions were shown (PRMT5/miR145; SRSF6/miR375; RBMX/miR23a; RBMX/miR24; RBMX/miR125a5p; SRSF11/miR143).Using the "Luciferase Reporter Gene Assay" technique coupled with site directed mutagenesis, we have shown that miR145 negatively regulated the expression of PRMT5 by interaction with its 3’untranslated translated region. Due to the control of the premessenger RNA splicing machinery by PRMT5, we propose that miR145 could be a general splicing regulator.

Filip Lhota: Study of alternatively spliced variants of estrogen receptor alpha in breast cancer cell lines Abstract: Estrogen receptor α (ER-α) is a transcription factor responsible for mediation of the activities of its natural ligand 17-β-estradiol (E2), the hormone that together with progesterone belongs to the key regulators of mammary epithelial as well as breast cancer cells proliferation. Except to the major gene product consisting of all eight coding exons of ER-α, numerous qualitatively and quantitatively different spliced variants originated from primary transcript by activity of alternative splicing is expressed. Despite that some of these spliced variants have been functionally characterized, their precise role on final ER-α cellular activity remains to be elucidated. The functional characterization of individual alternative forms of ER-α and description of its participation on the overall ER-α activity is important for our understanding of their biogenesis and is also critical for the delineation of molecular bases for ER-α regulation during anti cancer chemotherapy. This work aimed to study the influence of alternatively spliced ER-α variants on the growth characteristics of clones constructed from stable mammary tissue cell lines in regulation to cultivation conditions and cellular...

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Precise engineering of genomes holds great promise to advance our understanding of gene function and biotechnological applications. DNA double strand breaks are repaired via imprecise non-homologous end joining repair or via precise homology-directed repair processes. Therefore, we could harness the DSBs to engineer the genomes with a variety of genetic outcomes and with singlebase- level precision. The major barrier for genome engineering was the generation of site-specific DNA DSBs. Programmable DNA enzymes capable of making a complete and site-specific cut in the genome do not exist in nature. However, these enzymes can be made in in vitro as chimeric fusions of two modules, a DNA binding module and a DNA cleaving module. The DNA cleaving module can be programmed to bind to any user-defined sequence and the DNA cleaving module would generate DSBs in the target sequence. These enzymes called molecular scissors include zinc finger nucleases (ZFNs) and transcriptional activator like effector nucleases (TALENs). The programmability of these enzymes depends on protein engineering for DNA binding specificity which may be complicated, recourse intensive and suffer from reproducibility issues. Recently, clustered regularly interspaced palindromic repeats (CRISPR)/ CRISPR associated endonuclease 9 (Cas9) an adaptive immune system of bacterial and archaeal species has been developed for genome engineering applications. CRISPR/Cas9 is an RNA-guided DNA endonuclease and can be reprogrammed through the engineering of single guide RNA molecule (sgRNA). CRISPR/Cas9 activity has been shown across eukaryotic species including plants. Although the engineering of CRISPR/Cas9 is quite predictable and reproducible, there are many technological challenges and improvements that need to be made to achieve robust, specific, and efficient plant genome engineering. Here in this thesis, I developed a number of technologies to improve specificity, delivery and expression and heritability of CRISRP/Cas9-modification in planta. Moreover, I used these technologies to answer basic questions to understand the molecular underpinning of the interplay between splicing and abiotic stress. To improve Cas9 specificity, I designed and constructed a chimeric fusion between catalytically dead Cas9 (dCas9) and FOKI catalytic DNA cleaving domain (dCas9.FoKI). This synthetic chimeric fusion enzyme improved Cas9 specificity which enable precision genome engineering. Delivery of genome engineering reagents into plant cells is quite challenging, I developed a virus-based system to deliver sgRNAs into plants which facilitates plant genome engineering and could bypass the need for tissue culture in engineering plant genomes. To improve the expression of the CRISPR/Cas9 machinery in plant species, I developed a meiotically-driven expression of CRISPR/Cas9 which improved genome editing and heritability of editing in seed progeny, thereby facilitating robust genome engineering applications. To understand the molecular basis of the interplay between splicing stress and abiotic stress, I used the CRISPR/Cas9 machinery to engineer components of the U2snRNP complex coupled which chemical genomics to understand the splicing stress regulation in response to abiotic stress conditions. Finally, I harnessed the technological improvements and developments I have achieved with CRISPR/Cas9 system to develop a directed evolution platform for targeted trait engineering which expands and accelerates trait discovery and engineering of plant species resilient to climate change conditions.

RNA provides the framework for the assembly of some of the most intricate macromolecular complexes within the cell, including the spliceosome and the mature ribosome. The assembly of these complexes relies on the coordinated association of RNA with hundreds of trans-acting protein factors. While some of these trans-acting factors are RNA binding proteins (RBPs), others are adaptor proteins, and others still, function as both. Defects in the assembly of these complexes results in a number of human pathologies including neurodegeneration and cancer. Here, we demonstrate that Silencing Defective 2 (SDE2) is both an RNA binding protein and also a trans-acting adaptor protein that functions to regulate RNA splicing and ribosome biogenesis. SDE2 depletion leads to widespread changes in alternative splicing, defects in ribosomal biogenesis, and ultimately complete loss of cell viability. Our data highlight SDE2 as a previously uncharacterized essential gene required for the assembly and maturation of some of the most fundamental processes in mammalian cells.

Alternative splicing of the zebrafish Myosin Phosphatase Targeting Subunit, MYPT1, produces a novel isoform (TV202). TV202 and the truncated TV202Δ ere shown to form an active complex with Protein Phosphatase 1 β (PP1β) via stress fiber assay. TV202 was also shown to be localized in the cytoplasm, enriched in a paranuclear manner. TV202Δ was found the be localized inside the nucleus. It was also found that TV202 was zygotically, but not maternally, expressed during early zebrafish development via RT-PCR.

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