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This book was written for graduate and medical students, as well as clinicians and postdoctoral researchers. It describes the theory of alternative pre-mRNA splicing intwelve introductory chapters and then introduces protocols and their theoretical background relevant for experimental research. These 43 practical chapters cover: Basic methods, Detection of splicing events, Analysis of alternative pre-mRNA splicing in vitro and in vivo, Manipulation of splicing events, and Bioinformatic analysis of alternative splicing.A theoretical introduction and practical guide for molecular biologi

RNA splicing. --- Alternative Splicing. --- RNA Precursors. --- RNA Precursors --- Alternative splicing. --- Rna precursors. --- Rna splicing. --- Messenger RNA.

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Life --- RNA splicing --- RNA Splicing. --- Transcriptome. --- Humans. --- Origin

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Life --- RNA splicing --- RNA Splicing. --- Transcriptome. --- Humans. --- Life --- RNA splicing. --- Origin --- Origin.

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ln this work , we aimed to develop novel tools for the analysis of function and biogenesis of micrq-RNAs . Since we worked on two different methods, this work is divided in two parts.The aim of the first part was to develop a quick, reliable and cheap method to synthesize and purify precursor-microRNAs (premiR). Our method is based on in vitro transcription by T7 RNA polymerase. To overcome limitations of this technique, we added special RNA structures (ribozymes) capable of cleaving the transcript in an autocatalytic manner and leaving only the desired premiR sequence. To be able to extract the ribozymes, which could interfere with further processing of the premiR, we added a second RNA structure (aptamer) capable of binding to an affinity matrix. Eventually, we were able to synthesize and purify different premiRs that could be cleaved by the enzyme Dicer.The second part of this thesis focused on the inhibition of micro-RNA. There already are different methods to inhibit them but they all suffer serious limitations. That's why we attempted to improve the method of the so-called micro-RNA sponges. These are transcr ipt containing many binding sites for a miR. These transcripts act as compet itive inhibitors by trapping the micro-RNA. However, these sponges are far from efficient due to the destabilization caused by the binding of the micro-RNA. This destabilization is caused by the decapping and deadenylation of the transcript. We therefore proposed that, if we were able to produce circular sponges these would probably be more stable and hopefully more efficient. To explore this hypothesis, we first tried to find a model allowing us to generate circular miRNA sponges. Three different experimental were generated and assessed for their capacity to generate circular sponge transcripts. Furthermore, we tested whether the best of the experimental systems could be integrated into a lentiviral vector that would allow us to infect a variety of cells.Future work will have to demonstrate whether the generated circular sponges are more efficient inhibitors of miRNA function when compared to their linear counterparts . Au cours de ce mémoire, nous nous sommes intéressés au développement de nouveaux outils permettant l'analyse de la fonction et de la biogenèse des micro-ARNs . Dans cette optique, nous avons exploré deux méthodes différentes. Par conséquent , ce travail est scindé en deux parties.Le but de la première partie de ce mémoire était le développement d'une méthode de synthèse de micro-RNA précurseur (premiR). A insi, nous avons mis au point une méthode sur base de la transcription in vitro. Afin d'éviter certaines contraintes du point de vue de la séquence , nous avons flanqué le premiR d'une séquence ARN particulière (ribozyme) capable de cliver tout nucléotides supplémenta ire éventuels. Ensuite, afin d'être capable d'extraire ces séquences contaminantes, nous avons complémenté les ribozymes d'une seconde structure ARN (aptamères) capable de se lier avec une matrice d'affinité. Suite à la transcription in vitro et la purification du premiR, nous avons analysé le résultat par électrophorèse en gel de polyacrylamide dénaturant et « Northern Blot ». Nous avons pu démontrer l'efficacité de notre méthode à produire des premiR, les purifier ainsi que montrer l'utilité de ceux-ci comme outils pour étudier le clivage catalysé par Dicer.La seconde partie de ce mémoire se penche sur le développement d'une nouvelle méthode d'inhibition des miRs. En effet, malgré l'existence d'outils permettant leur inhibition, ceux-ci souffrent encore de nombreux désavantages . Nous avons donc tenté d'améliorer la méthode d'éponges à miR décr ite précédemment dans la littérature. Cependant, afin d'améliorer leur stabilité, nous avec tenté de produire des éponges circulaires. Pour cela nous avons complémenté notre construction de signaux d'épissage et de séquences répétitives nécessaires à la circularisation comme décrit dans la littérature. Après transfection de nos constructions plasmidiques, nous avons mesuré les niveaux d'ARN circulaires et linéaires à l'aide de PCR quantitative . Ensuite nous avons développé des vecteurs lentiviraux permettant l'intégration stable de ces constructions. De futures études devront démonter si le système ainsi développé présente des améliorations par rapport aux autres techniques.

MicroRNAs --- RNA Splicing --- RNA --- Transcription, Genetic

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This eBook is a collection of articles from a Frontiers Research Topic. Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: frontiersin.org/about/contact

splicing factor --- development --- stress --- adaptation --- evolution --- environment --- flowering --- germination