PROCESSING AND FUNCTION OF POLYOMA RNA

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The oncogenic polyoma mouse virus provides us with a good model system for studying not only the molecular biology of cell transformation and tumorigenesis, but also mechanisms of regulation of eukaryotic gene expression. Our primary interests are in better understanding how viral RNA molecules are made and processed in infected cells, with a major focus on late-strand RNAs. Our particular interests are the structure and regulation of the late promoter, late pre-MRNA processing, and the switch from mostly early-strand MRNAS before the initiation of viral DNA synthesis to mostly late-strand MRNAS afterwards. The major project will be to dissect the late promoter both in vivo and in vitro. This promoter belongs to the initiator class of promoters. Specific sequences that are important for its function will be determined, and factors regulating its activity will be identified, purified and cloned. Late viral RNA processing involves exon skipping, as well as alternative selection of 3' splice sites. The mechanism by which splice site use is regulated will be determined through the study of numerous mutants and double- genome constructs. Genetic techniques will also be used to learn how some unspliced late MRNAS accumulate in the nucleus and cytoplasm of infected cells. Finally, studies will be carried out to learn what regulates the early-late switch. Effects of DNA replication, large T antigen, promoter sequences and polyadenylation site sequences on the relative accumulation of early-strand and late-strand RNAs will be determined. Various models that can explain the switch will be tested, including one in which early-strand RNA is downregulated at late times by the production of large amounts of giant late-strand transcripts that form antisense hybrids with them.