Polyoma virus will be used as a model system to study the synthesis and processing of mammalian RNA molecules. We have three major specific aims.
The first aim i s to perform a number of studies designed to understand the switch from mostly early-strand mRNAs before the initiation of viral DNA synthesis to mostly late-strand mRNAs afterwards. The regulation of both early and late RNA levels is not at the promoter level, and is post- transcriptional. The regulation of early-strand gene expression is by a novel mechanism (nuclear antisense RNA from the late strand) which is likely to be quite generally used by cells to regulate the expression of many of their own genes. This virus provides a powerful genetic and biochemical system in which to study antisense regulation and to learn how to exploit this knowledge to regulate the expression of other genes, both viral and cellular. We have shown that many nuclear early-strand RNAs are modified by the enzyme double strand RNA adenosine deaminase in the presence of late-strand antisense molecules. This appears to be a major aspect of the downregulation of early gene expression at late times in the viral life cycle. We will follow the fate of these modified molecules, and investigate how they are formed. Late-strand RNA levels are regulated in a different way and appear to positively influence their own accumulation during infection. We will determine what sequences are critical for the activation of late gene expression after DNA replication initiation. In the second aim we will investigate several important and interesting aspects of late RNA processing. We will continue dissecting the late polyadenylation signal, both in vitro and in vivo, especially to identify and characterize a putative cell factor that interacts with a recently recognized hexanucleotide element repeated three times in the minimal poly(A) signal region. In related work, we will develop new systems to investigate the relationship between polyadenylation and transcription termination. Also, we have planned a number of experiments to test various hypotheses about the nature of nucleocytoplasmic transport of unspliced polyoma late messages. For our third aim we will continue our studies on the structure and function of the late promoter. We have identified sequences spanning a major late transcription startsite as important for transcription initiation and have cloned a cellular factor that interacts with them. The Kruppel-related factor we have cloned will be characterized better, and we will begin a more detailed analysis of its biochemical function in polyoma late transcription initiation.
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