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Étude de la maturation de l'extrémité 3' non traduite et de la traduction de l'ARN messager codant pour l'histone H4.

Abstract : First we studied HBP protein interaction with the hairpin structure of histone H4-12 pre-mRNA. Starting from loss-of binding mutants of HBP, we selected by the yeast three hybrid system, HBP suppressors that restored the interaction for a wild-type hairpin RNA. Most of the selected mutations were located in both N- and C terminal domains of the protein, suggesting that these domains are involved in RNA recognition. Next we analyzed the structural effect of HBP binding to the 3'UTR of several histone pre-mRNA. Structural probing revealed strong secondary structures in the 3'UTR molecules that could prevent U7 snRNP anchoring to the RNA. We also showed that in some cases HBP-binding induced conformational changes at the level of U7 hybridization sequence. These changes were associated to a better annealing of a U7 snRNA transcript. However, this mechanism is not general to all histone-genes. Lastly, we focused on histone translation mechanism. We found that in vitro translation of H4-12 mRNA is highly efficient. Surprisingly, we found that 5' and 3' UTR are dispensable, which suggests that the Open Reading Frame (ORF) alone is able to recruit the ribosome. Using enzymatic and chemical probing we solved the secondary structure of the whole mRNA. The molecule appears to be circular due to hybridization of 5' and 3' sequences. With non-sense RNA designed to inhibit translation we identified essential nucleotides required for a highly efficient translation mechanism. Altogether, the data from in vitro translation, mutagenesis, toe print and electron-microscopy led us to propose a model that explains the highly efficient mechanism of histone translation. First, the ORF might efficiently recruit two ribosomes on Internal Ribosome Entry Sites (IRES). Second, after translation of the histone circular histone-mRNA an efficient recycling process would channel the ribosomes onto the initiation-codon for a new round of translation.
Keywords : human HBP protein, H4 histone mRNA, three-hybrid system, genetic selections, structural RNA probing, 3' end processing, U7 snRNP, in vitro translation , IRES, structure-function studies.
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Submitted on : Friday, January 26, 2007 - 3:48:10 PM
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  • HAL Id : tel-00126923, version 1



Sophie Jaeger. Étude de la maturation de l'extrémité 3' non traduite et de la traduction de l'ARN messager codant pour l'histone H4.. Biochimie [q-bio.BM]. Université Louis Pasteur - Strasbourg I, 2005. Français. ⟨tel-00126923⟩



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