Splicing of mRNA precursors (pre-mRNA) comprises a series of ATP-dependent steps, the first of which is the stable binding of U2 snRNP at the pre-mRNA branchpoint. The basis of ATP use in splicing is not well understood. Several yeast splicing factors belong to DEAD/H box family of RNA-dependent ATPase, and are implicated in dynamic RNA structure rearrangement during spliceosome assembly. In mammals, however, such information is conspicuously lacking. In fact, none of the known mammalian splicing factors has characteristics for ATP hydrolysis. In an attempt to identify mammalian splicing factors involved in ATP usage, we have developed a novel approach to identify and purify spliceosomal ATP binding proteins. Six spliceosomal ATP binding proteins were found, one of them, SAFp56, was purified and microsequenced, and found to be a DEAD box protein containing unique DECD motif instead of the canonical DEAD motif. During the course of this work, a new functional region in U2AF65, an essential splicing factor required for U2 snRNP entry into the spliceosome, was defined. This information was used to clone a human U2AF65 associated protein (UAP). UAP and SAFp56 are identical. We refer to this protein as hUAP56 (human 56 kDa U2AF65 associated protein). We present evidence that hUAP56 is an essential splicing factor required for the U2 snRNP binding to pre-mRNA. Interestingly, UAP56 is recruited to pre-mRNA in a polypyrimidine tract bound U2AF65-dependent fashion. This result underscores a new function of U2AF65, and provides the first description of how a specific DEAD box protein is directed to a pre-mRNA splicing signal, and/or, to the proximity of its substrate at a particular stage. Like an authentic DEAD box protein. hUAP56 has ATP binding, RNA-stimulated ATPase, as well as RNA binding activity. A particularly novel result is that the ATPase activity of UAP56 is stimulated by U2AF65. This observation strongly suggests the role of UAP56 in ATP dependent mechanism during U2 snRNP binding to the pre-mRNA branchpoint, and implies that UAP56 may function through a distinct mechanism. We identify yeast UAP (yUAP), a highly conserved S. cerevisiae homologue of hUAP56. yUAP is essential for viability, can be functionally substituted for by hUAP56, and like its human counterpart, is an essential pre-mRNA splicing factor required for spliceosome assembly. Furthermore, we show that yUAP is required for formation of the branchpoint dependent commitment complex, the precursor for U2 snRNP addition. Site-directed mutagenesis revealed that all DEAD box protein consensus motifs are required for yUAP function. Interestingly, a strain harboring a yUAP mutant in which the DECD sequence, characteristic of UAP members, was changed to canonical sequence, is inviable. Our results demonstrate that UAP is structurally and functionally conserved from yeast to man. In conjunction with previous studies, we conclude that at least two DEAD box proteins, Prp5p and yUAP, are required for the U2 snRNP-branchpoint interaction.

RNA molecules are diverse in form and function. They include messenger RNAs (mRNAs) that are templates for proteins, splice products such as introns that can generate functional noncoding RNAs, and a slew of smaller RNAs such as transfer RNAs (tRNAs) that help decode mRNAs into proteins. RNAs can show distinct patterns of subcellular localization that play an important role in protein localization. However, RNA distribution in cells is incompletely understood, with prior studies focusing primarily on RNAs that are long (>200 nucleotides), fully processed, and polyadenylated. We examined the distribution of RNAs in neurons. Neuronal compartments can be separated by long distances and play distinct roles, raising the possibility that RNA localization is especially overt and functionally meaningful in these cells. In our exploration, we physically dissected projections from cell bodies of neurons from the rat brain and sequenced total RNA. We describe two main findings. First, we identified excised introns that are enriched in neuronal projections and confirmed their localization by single- molecule fluorescence in situ hybridization. These are a previously unknown set of circular RNAs in neuronal projections: tailless lariats that possess a non- canonical C branchpoint. Second, we observed a highly abundant population of small (20-150 nucleotide) RNAs in neuronal projections, most of which are tRNAs. For both circular introns and tRNAs, we did not observe known RNA localization signals. Thus, many types of RNA, if sufficiently stable, appear free to diffuse to distant locations, their localization perhaps aided by the movement of large organelles in the confines of neuronal projections. Our survey of RNA molecules across subcellular compartments provides a foundation for investigating the function of these molecules and the mechanisms that localize them.

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No description available.

Background: With the boom in the internet space and social media platforms, a large number of images are being shared. With this rise and advancements in technology, many image editing tools have made their way to giving rise to digital image manipulation. Being able to differentiate a forged image is vital to avoid misinformation or misrepresentation. This study focuses on the splicing image forgery to localizes the forged region in the tampered image. Objectives: The main purpose of the thesis is to extend the capability of the JPEG Ghost model by localizing the tampering in the image. This is done by analyzing the difference curves formed by compressions in the tampered image, and thereafter comparing the performance of the models. Methods: The study is carried out by two research methods; one being a Literature Review, whose main goal is gaining insights on the existing studies in terms of the approaches and techniques followed; and the second being Experiment; whose main goal is to improve the JPEG ghost algorithm by localizing the forged area in a tampered image and to compare three machine learning models based on the performance metrics. The machine learning models that are compared are Random Forest, XGBoost, and Support Vector Machine. Results: The performance of the above-mentioned models has been compared with each other on the same dataset. Results from the experiment showed that XGBoost had the best overall performance over other models with the Jaccard Index value of 79.8%. Conclusions: The research revolves around localization of the forged region in a tampered image using the concept of JPEG ghosts. This is We have concluded that the performance of XGBoost model is the best, followed by Random Forest and then Support Vector Machine.

La maladie de Huntington (MH) est une maladie autosomale dominante causée par une expansion de la répétition CAG codant pour une expansion de la polyglutamine dans le premier exon du gène Huntingtine (HTT). Ce gène code pour une protéine ubiquitaire dont la mutation entraine de graves symptômes moteurs, psychiatriques et cognitifs, dus à la dégénérescence spécifique des neurones GABAergique épineux moyens du striatum. Nous proposons d'utiliser le trans-épissage pour développer un vecteur de thérapie génique qui réduira significativement voir éliminera l'expression de la protéine mutée tout en restaurant un niveau physiologique de HTT normale dans les cellules affectées par la mutation du gène Huntingtine. Cette technologie est basée sur le remplacement de l'exon muté par un exon sans mutation pendant l'étape de maturation de l'ARNm. Du fait du caractère dominant de la mutation,l'efficacité thérapeutique nécessitera une réaction de trans-épissage très efficace capable de convertir une portion significative de pre-ARNm HTT mutés en en ARNm HTT normaux. Nous avons donc développé un système rapporteur fluorescent permettant la détection des évènements de trans-épissage afin d’identifier les séquences les plus performantes parmi une centaine de molécules candidates. Nous avons validé notre stratégie de criblage basée sur la fluorescence et réalisé le criblage sur plusieurs introns HTT (3, 9 et 20) qui ont démontré des zones favorables au trans-épissage. Une méthode de quantification directe et absolue du taux de trans-épissage a également été validée pour déterminer très précisément le taux de correction. L’ensemble de ce travail a permis de contribuer à la mise en évidence de la faisabilité du trans-épissage dans le contexte de la MH. / Huntington’s disease (HD) is an autosomal dominant genetic disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in the first exon of the Huntingtin gene (HTT). This gene encode a ubiquitous protein in which mutation lead to severe motor, psychiatric and cognitive deficits and causes degeneration of specific neuronal populations, in particular the GABAergic medium spiny neurons of the striatum. We propose to use trans-splicing to develop a gene therapy vector that will significantly reduce or eliminate the expression of the mutant protein while restoring a physiological level of normal HTT in cells affected by the HD mutation. This technology is based on replacement of the mutated exon by a normal version during the mRNA maturation process. HTT mutation being dominant, therapeutic benefits necessitates a highly efficient trans-splicing reaction that would convert a significant proportion of mutant-HTT pre-mRNA into normal HTT mRNA. For this purpose, we developed a fluorescent reporter system enabling the detection of trans-splicing events in high content screening in order to identify the most potent trans-splicing sequences among hundreds of molecules. We validated our fluorescent screening strategy and implement trans-splicing screening on 3 HTT introns (3, 9 and 20), in which we demonstrated the presence of hotspot promoting trans-splicing reactions. A direct and absolute quantification method was also validated to accurately assess the correction rate. Overall, this work generated additional evidences of trans-splicing feasibility in HD.

La voie de signalisation mTOR intègre une variété de signaux environnementaux pour réguler la croissance et le métabolisme cellulaire. Cette voie est altérée dans 70% des cancers. Les inhibiteurs allostériques de mTOR, comme la rapamycine et ses dérivés (évérolimus et temsirolimus), sont administrés aux patients atteints de tumeurs métastatiques du sein, du rein et neuroendocrines. Cependant, leur efficacité reste modeste et une majorité de patients rechutent. L'utilisation de rapalogues fait donc face à deux problèmes cliniques majeurs : 1/l’absence de biomarqueur qui permette de stratifier les patients qui bénéficieraient le plus d'un traitement par rapalogues ; 2/ l’existence de plusieurs mécanismes de résistance décrits ou non. L’objectif de mon travail de thèse est d’identifier des nouveaux gènes cible des rapalogues utilisables comme biomarqueurs prédicteurs de l’efficacité du traitement ou comme cibles thérapeutiques pour vaincre la résistance.Nous avons identifié le gène TRIB3 comme cible des rapalogues. Sous traitement, son expression est diminuée dans un panel de lignées tumorales et des échantillons tumoraux. Nous avons démontré que cette régulation est indépendante de l’inhibition de la voie mTOR, mais médiée par le répresseur transcriptionnel GCF2. Des analyses protéomiques à haut débit ont identifié TRIB3 en tant que composant du spliceosome. De plus, nous avons démontré que la régulation négative de TRIB3 est nécessaire aux rapalogues pour modifier l'épissage des pré-ARNm. A l’inverse, la surexpression de TRIB3 supprime ces effets des rapalogues. En conclusion, ce travail de thèse ouvre plusieurs perspectives: 1 / l'utilisation potentielle de TRIB3 comme biomarqueur pour prédire ou évaluer l'efficacité du traitement par les rapalogues; 2 / de nouvelles opportunités thérapeutiques ciblant ces mécanismes indépendants de mTor ; 3/ la combinaison possible des rapalogues avec des composés ciblant l’épissage afin de surmonter la résistance. / The mTOR signaling pathway senses variety of environmental cues and integrates them to regulate cellular growth and metabolism. This pathway is altered in 70% of cancers. Allosteric inhibitors of mTOR like rapamycin and its derivatives (everolimus and temsirolimus) have become standard of care in patients with metastatic breast, kidney and neuroendocrine tumors. Unfortunately, their role is modest and most of patients will relapse. Thus, in clinic there are two major concerns related to the use of rapalogs: 1/ the absence of accurate biomarker to stratify patients who would benefit from rapalogs treatment; 2/ the existence of known and unknown mechanisms of resistance. Accordingly, the aim of my PhD project is to identify new target genes of rapalogs that could be used as biomarkers to predict treatment efficacy, or as therapeutic targets, to overcome resistance.We identified TRIB3 gene as a novel target of rapalogs. Upon treatment, its expression is down-regulated both in a panel of cancer cell lines and in cancer patient samples. We showed that this regulation is independent of the mTOR signaling inhibition, but relies on a transcriptional regulation via the co-repressor GCF2. High-throughput proteomic analyses identified TRIB3 as a component of the spliceosome. Additionally, we demonstrated that the down-regulation of TRIB3 is necessary for rapalogs to alter pre-mRNA splicing. In contrast, the, overexpression of TRIB3 abolishes these effects of rapalogs. In conclusion, this PhD work leads to the following important perspectives: 1/ the potential use of TRIB3 as a biomarker to predict or asses the efficacy of rapalogs treatment; 2/ new window of therapeutic possibilities by targeting this mTOR - independent mechanism of action; 3/ the potential combination of rapalogs with splicing targeting agents to overcome resistance.

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.

No description available.

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.