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Étude des dérégulations de l'épissage alternatif du pré-ARN messager de la troponine T cardiaque humaine associées aux dystrophies myotoniques de types 1 et 2 et des caractéristiques du facteur d'épissage MBNL1 impliqué dans ces pathologies

Abstract : Amplifications of CTG motifs in the human DMPK gene are responsible for Myotonic Dystrophy of type 1. The resulting CUG repeats in pre-mRNAs capture the MBNL1 splicing factor, leading to mis-regulation of MBNL1 pre-mRNA targets. Due to the recent discovery of MBNL1 and its numerous isoforms (9) resulting from alternative splicing, little is known on how MBNL1 regulates splicing and how a decreased level of available MBNL1 generates splicing miss-regulations. First, we defined which of the MBNL1 alternative and constitutive exons are required for: i) RNA binding, ii) splicing activity and, iii) MBNL1 sub-cellular localization. Second, for a more precise definition of the MBNL1 RNA binding properties, we performed SELEX experiments using a library of RNA stem-loop structures containing a 18-nt long randomized sequence. Its leads to the identification of 12-nt long sequence adopting a peculiar stem-loop structure, whose importance for MBNL1 binding was revealed by its preservation by compensatory base-pair mutations. Finally, based on the above data, we studied the mechanisms involved in regulation of hcTNT exon 5 splicing. By in cellulo assays, we defined the hcTNT pre-mRNA region required for both normal inclusion and for the trans-dominant effect of CUG repeats. Within this region, we identified six new potential MBNL1 sites and demonstrated their functional role by in vitro and in cellulo assays. We also identified several additional splicing regulatory elements involved in normal and CUG-deregulated exon 5 inclusion and already showed a role of hnRNP H in splicing regulation. Altogether, our data bring new information important for understanding the pathology
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https://hal.univ-lorraine.fr/tel-01746306
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  • HAL Id : tel-01746306, version 1

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Audrey Vautrin. Étude des dérégulations de l'épissage alternatif du pré-ARN messager de la troponine T cardiaque humaine associées aux dystrophies myotoniques de types 1 et 2 et des caractéristiques du facteur d'épissage MBNL1 impliqué dans ces pathologies. Médecine humaine et pathologie. Université Henri Poincaré - Nancy 1, 2011. Français. ⟨NNT : 2011NAN10141⟩. ⟨tel-01746306⟩

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