Development of a method to identify RNA-binding proteins:Study of the transcription and translation mechanisms among Schmallenberg virus

Résumé

After genornic sequencing analysis, Schmallenberg virus was categorized as a member of Bunyaviridae family, the Orthobunyavirus genus and the Simbu serogroup. Its tripartite genome is composed of negative sense RNA segments (L, M and S), encoding 6 proteins in total. These genornic segment, consisting in a coding region flanked by 3' and 5' UTR, are transcribed into mRNAs, constituted of a 5' host-derived sequence and lacking a poly(A) tail at the 3' end. However, poly(A) tail is crucial for the mRNA stability, the transcription termination and the mRNA translation initiation processes. In silico analyses recently revealed a conserved stem-loop and a putative transcription termination signal both localized in the 3' UTR of the SBV antigenomic S segment, indicating a potential functional relevance in viral replication. Our objective was to develop an RNAaffinity purification technique coupled with mass spectrometry analysis (RAP-MS) to identify
the RNA-binding proteins interacting with these two elements. In brief, the RNA bait, containing both elements of interest, was immobilized on a solid support, in a reversible manner. As the virus is described to replicate in the cytoplasm,
cytosolic extracts were incubated with the immobilized RNA. After washing steps to eliminate non-specifically bound proteins, the ribonucleoproteins complexes were specifically eluted from the solid matrix and subjected to mass spectrometry. Although some results suggest that the conditions settled for protein capture on the RNA are not optimal, several proteins described to play a role in viral transcription/ translation/ replication processes identified by RAP-MS. However, these results have to be taken with caution since they are based on only one experiment. Therefore, the RAP-MS experiment has to be repeated with independent replicates. If successful, this technique would confirm the presumed role of the two conserved elements in SBV transcription/translation processes and would highlight the proteins contributing to those mechanisms. More generally, it should provide further insight on transcription termination and translation initiation processes in the absence of poly(A) tail

la date de réponse	2016
langue originale	Anglais
L'institution diplômante	Universite de Namur
Superviseur	Patricia Renard (Promoteur)