A proteomic approach for studying LTR retrotransposon host factors in saccharomyces cerevisae
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The Ty retrotransposons of S. cerevisiae are mobile elements that resemble retroviruses, both in their genetic structures and replication cycles. Ty retrotransposition begins in the nucleus with the transcription of Ty RNA. Following translation of Ty proteins, virus-like particles (VLPs) assemble in the cytoplasm. VLPs contain Ty RNA and enzymes surrounded by a capsid shell. Specific cellular tRNAs are also packaged within VLPs and serve as primers for reverse transcription of Ty element RNA into dsDNA. Ty dsDNA enters the nucleus and becomes inserted into the cellular genome, either by integrase-mediated integration or by homologous recombination, completing a cycle of retrotransposition. A wide range of cellular host factors involved in the Ty replication cycles has been identified by genetic screens; however little is known about the mechanism by which most of these affect Ty element replication. To complement these genetic approaches we have identified the host proteins associated with Ty1 VLPs by performing affinity purification of VLPs followed by mass spectrometric analyses. Almost 100 host proteins are associated with Ty1 VLPs. This list only minimally overlaps with the lists generated by previous genetic screens for host factors and includes many essential host proteins. As with the previous genetic screens, the challenge is to determine which VLP-associated host proteins play a sensible biological role in retrotransposition. Proteins that have also appeared in other screens are obvious candidates for further study. In addition, we hypothesize that there is an increased probability of identifying host proteins that play a distinguishable role in retrotransposition among the subgroup that is specifically packaged into VLPs. In chapter 1, I describe my discovery that two RNA binding proteins, Puf6p and Khd1p, play roles in Ty1 RNA localization and I produce evidence that supports the idea of a “specialized ribosome”. In chapter 2, I describe my analysis of a population of uncapped Ty1 RNA in VLPs. I also describe experiments on the role of the RNA lariat debranching enzyme Dbr1p in Ty1 retrotransposition, including my work establishing the association of Dbr1p with Ty1 VLPs.
Table of Contents
Association of Puf6p and Khd1p with VLPs of the yeast retrotransposon TY1 -- Association of the debranching enzyme Dbr1p with VLPs of the yeast retrotransposon TY1 -- Materials and methods
Cell Biology and Biophysics and Molecular Biology and Biochemistry