Post-transcriptional regulation of the processing of eukaryotic pre-mRNA (splicing, polyadenylation, editing, etc...) is an important control point in gene expression in eukaryotes. We recently discovered a novel form of gene regulation where the U1A protein, a component of the U1 snRNP involved in pre-mRNA splicing, negatively autoregulates its own production by binding to and inhibiting the polyadenylation of its own pre-mRNA (38- 41). The complex between U1A and the U1A pre-mRNA binds to and blocks the activity of polyA polymerase (PAP), the enzyme which catalyzes addition of the polyA tail. U1A autoregulation is the first example of its kind. As part of the original proposal we characterized another system in which the U1 snRNA-associated 70K protein inhabits polyadenylation by also inhabiting PAP. Unlike the case of U1A, however, 70K inhibits PAP while being indirectly bound to the pre-mRNA through the U1 snRNP complex which is base-paired to the pre-mRNA. Surprisingly, even though U1A is present in the U1 snRNP complex, the PAP-inhibitory activity resides entirely with the 70K protein. The discovery that the polyadenylation inhibitory motifs in both U1A and 70K are conserved and are found in other proteins suggests that polyadenylation regulation via PAP inhibition will be more widespread than previously thought. The framework for this proposal is to use the examples of U1A and 70K as a basis to achieve the following goals which are: 1) to determine how prevalent inhibition of PAP is as a regulatory mechanism by finding additional examples, 2) to characterize the cis- and transacting sequence motifs in order to define a family of proteins able to control gene expression via inhibition of PAP, and 3) to determine whether this type pf regulation can be used to control expression of heterologous genes in vivo. The achievement of this proposal will advance fundamental knowledge of pre-mRNA processing and indicate the generality of regulation of polyadenylation by U1 snRNA-associated proteins. By elucidating the molecular mechanisms by which these two splicing-associated proteins control the polyadenylation machinery, we will provide insight into the competition between splicing and polyadenylation that in many cases forms the basis for how alterative processing of pre-mRNAs is governed.
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