Post-transcriptional regulation of the processing of eukaryotic pre-mRNA (splicing, polyadenylation, editing etc.) is an important control point in gene expression in eukaryotes. For nearly all mRNAs, the polyA tail is essential for translation, stability, and nuclear export. Changes in the position of the polyA tail cause changes in the final mRNA sequence leading to additional regulatory possibilities. Regulation of polyA sites, either by alternative polyA site choice or by an """"""""on-off"""""""" switch of a single polyA site, was generally considered rare. Remarkably, it is now estimated that 30% of human genes are expressed as mRNAs with different polyA sites, a number likely to be applicable to other vertebrates, as the human genome and EST database are the most complete and best annotated of the vertebrates. Even if only a fraction of this 30% represents regulated polyA sites, we are likely to see a dramatic upward revision in the frequency and importance of this form of regulation. In the first funding period of this grant we: 1) characterized a unique form of polyA tail regulation by the U1A splicing factor, 2) discovered that U1A, via binding non-consensus pre-mRNA sequences, can regulate polyA tail addition to genes other than just to itself and that other proteins are also capable of regulating polyA tail addition in a similar manner, and 3) demonstrated that U1 snRNP can be used as a tool to specifically inhibit expression of specific endogenous genes by inhibiting polyA tail addition. The framework of this proposal builds on work from the first funding period to achieve the following goals: 1) improve/characterize the U1in technique thereby allowing us and others to exploit it to its full potential, 2) investigate a new type of polyA tail regulation involving naturally occurring examples of U1 snRNP inhibiting expression of endogenous genes, and 3) identify/characterize new genes targeted for polyA tail regulation by U1A or by proteins previously shown to have the ability to regulate polyA tail addition. The achievement of these goals will advance fundamental knowledge of mechanisms of regulated gene expression, fully develop a promising new gene inhibitory technique, and elucidate the functional consequences for how components of the splicing machinery nteract with and regulate the polyadenylation machinery.
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