Hyjazie, Huda (2011). Factors involved in alternative polyadenylation during herpes simplex virus 1 infection Mémoire. Québec, Université du Québec, Institut National de la Recherche Scientifique, Maîtrise en virologie et immunologie, 101 p.
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Herpes simplex virus 1(HSV-1} is an important human pathogen that infects more than 80% of the population and causes serious disease in immunocompromised individuals and newborns. This virus has a dense DNA genome that codes for more than 80 genes, thus necessitating effective genetic regulation. One of the modes of regulation utilized by HSV-1is alternative polyadenylation: a post-transcriptional event that allows one gene to use two different polyadenylation signais (PAS}. This process generates transcripts that differ in their 3' untranslated region (3'UTR}, which can affect translation efficiency among ether things. Severa! HSV-1genes including UL24 and UL38 are regulated by alternative polyadenylation. UL24 short transcripts, which arise from the use of the UL24 polyadenylation signal, are expressed with early kinetics, while UL24 long transcripts, which arise from use of the UL26 polyadenylation signal, are expressed with late kinetics. We hypothesized that the switch in PAS usage is due to changes in the steady-levels of polyadenylation factors over the course of infection. We aIso hypothesized that the degree of association of the polyadenylation factors with the different polyadenylation sites changes over the course of the infection. ln order to test the first hypothesis, Vero and Hela cells were either mock-infected or infected for 3, 6, 9,12 and 16hrs,and cell lysates were prepared. We evaluated the steady-state levels of three subunits of polyadenylation factors,CPSF-100,CstF-64 and CFIm-25,by Western blot. We did not observe any reproducible change in the levels of these factors for the first 12 hours of infection, and therefore, a change in their levels could not explain the shift in PAS usage of the alternatively polyadenylated UL24 gene observed at around 10hpi. Severa subunits could not be evaluated using this technique due to commercial antibody availability and specificity. Nevertheless, though we did not observe a reproducible change in the steady-state levels of the factors tested during the first 12 hours of infection, we cannot rule out the possibility that the localization and the RNA or protein interactions of the various factors or subunits may have been affected. To test the second hypothesis, we had as an objective to identify proteins that interact with the 3'UTRs of alternatively polyadenylated transcripts during the infection. To identify these proteins,we developed an affinity purification system based on the S1RNA aptamer. We evaluated lysates from mock-infected cells as weil as cells infected for Shrs and 10hrs. Bound proteins were analysed by liquid chromatography-mass spectrometry (LC-MS}. Among other proteins, we identified eEFlA and NF45, both nuleic acid binding proteins. eEFlA plays a rote in translation fidelity and also assists in the nuclear export of mature tRNA, and NF45 is a transcriptional activator. These results serve as proof of principal for this strategy to identify proteins that interact with the 3'UTR of viral transcripts. Future work will be aimed at validating the proteins identified, and investigating other possible mechanisms involved in alternative polyadenylation such as post-translational modifications of polyadenylation factors. The results from this project will contribute to a better understanding of genetic regulation by alternative polyadenylation.
|Type de document:||Mémoire|
|Directeur de mémoire/thèse:||Pearson, Angela|
|Informations complémentaires:||Résumé avec symboles|
|Centre:||Centre INRS-Institut Armand Frappier|
|Date de dépôt:||30 nov. 2015 19:38|
|Dernière modification:||30 nov. 2015 19:38|
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