This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Saccharomyces cerevisiae contains five families of LTR retrotranspososns, Ty1-5, that are closely related to animal retroviruses. Like retroviruses, yeast Ty elements encode homologs of Gag and Pol proteins, form virus-like particles (VLPs), and transpose through RNA intermediates using reverse transcriptases to make Ty cDNAs. The RNAs of both retroviruses and LTR retrotransposons, grouped together as LTR retroelements, are more than passive intermediates;they are dynamic players in multiple steps of LTR retroelement replication, including promoting their own dimerization, packaging, and reverse transcription, and in some cases their own transcription and nuclear export. On top of all this, full-length LTR retroelement RNAs play two central roles: serving as element mRNAs, which are translated into proteins, as well as element genomic RNAs, which are packaged into virions or VLPs. How the RNA serves these two roles, either via segregated or interchangeable RNA populations, is only known in broad outline for a few LTR retroelements. Since LTR retroelements depend on their hosts to perform most of the functions necessary for their propagation, it is very likely that host factors are involved in most retroelement RNA dynamics, including the mechanism by which genomic and messenger RNA populations are distinguished. Genetic screens for host factors that play roles in Ty1 and Ty3 retrotransposition have identified hundreds of genes. However, little is known about how most of these host factors act in Ty element replication. To complement these genetic approaches we took a proteomic approach, identifying host proteins associated with Ty1 VLPs. We hypothesized that some of the host proteins physically associated with VLPs play direct (?proximal?) roles in retrotransposition, especially those proteins identified in previous, genetic screens for Ty host factors. We focused our attention on host proteins that might interact with Ty1 RNA and assist in keys aspects of Ty1 RNA dynamics. Our study involved affinity purification of Ty1 VLPs followed by mass spectrometric analysis (MudPIT). So far we have identified ~200 host proteins that specifically associate with Ty1 VLPs. Among these are Puf6p and Khd1p, two RNA binding proteins that inhibit translation of their target mRNAs. We have shown that puf6 and khd1 mutants are defective for Ty1 transposition and that at least Khd1p binds Ty1 RNA. We are currently exploring the roles of these proteins in Ty1 transposition.
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