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Étude de l'action sur l'épissage de protéines nucléaires se liant à la région de l'ARN du virus VIH-1 contenant le site d'épissage A7 et role de ces protéines sur d'autres sites accepteurs d'épissage de VIH-1

Abstract : HIV-1 pre-mRNA splicing depends upon 4 donor and 8 acceptor sites, which are used in combination to produce more than 40 different mRNAs. To further characterize nuclear factors involved in these processes, we purified RNP complexes formed by incubation of SLS2-A7 transcripts in HeLa cell nuclear extracts by affinity chromatography to identify new associated proteins. We showed that, in addition to the well known hnRNP A1 inhibitor of site A7, nucleolin, hnRNP H and hnRNP K interact directly with SLS2-A7 RNA. We demonstrated that hnRNP K has multiple binding sites in the vicinity of site A7 and that binds cooperatively to hnRNP A1 to the A7 RNA region and limits the A7 utilization in vitro. As hnRNP A1 is a negative regulator of several HIV-1 splicing sites (A1, A2, A3), we tested whether hnRNP K may also reinforce hnRNP A1 inhibition at these sites. Surprisingly, hnRNP K activated in vitro splicing of the D1-A1, D1-A2 and D1-A3 introns. Interestingly, hnRNP K was found to reinforce strongly the ASF/SF2 activity at site A2, which indicates that depending on the splicing site hnRNP K can be a splicing activator or inhibitor. To test how hnRNP K influences the relative utilization of HIV-1 splicing sites in cellulo, we used plasmid p PSP containing all the HIV-1 splicing sites and tested the effect of over-expression in HeLa cells on alternative splicing of the PSP RNA. Doubling the amount of hnRNP K in HeLa cells led to a drastic change of the PSP RNA alternative splicing, which confirms the strong influence of hnRNP K on alternative splicing. Moreover, increase of cellular concentration of hnRNP K strongly decrease the viral Nef protein production. hnRNP K protein affects A7 splicing regulation but also regulates the majority of regulated splicing sites of HIV. By extension of the study of hnRNP K effect to other HIV-1 splicing sites, we discovered that hnRNP K is a general regulator of HIV-1 splicing
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Maryline Santerre. Étude de l'action sur l'épissage de protéines nucléaires se liant à la région de l'ARN du virus VIH-1 contenant le site d'épissage A7 et role de ces protéines sur d'autres sites accepteurs d'épissage de VIH-1. Médecine humaine et pathologie. Université Henri Poincaré - Nancy 1, 2010. Français. ⟨NNT : 2010NAN10115⟩. ⟨tel-01746355⟩

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