The experiments described in this proposal are designed to provide insights into the mechanism and regulation of pre-mRNA polyadenylation, and how this process is linked to other nuclear events. The following four Specific Aims are proposed. 1. Regulation of poly(A) polymerase. Poly(A) polymerase (PAP) is regulated by phosphorylation and multiple isoforms can be generated by alternative splicing. These properties will be investigated by analysis of isogenic cell lines expressing wild-type PAP or a mutant defective in phosphorylation by cyclin-dependent kinases (cdk-PAP). The properties of a newly discovered PAP, called Neo-PAP, will be investigated. 2. Polyadenylation and RNA polymerase II. Studies of the function of RNAP II, and specifically the C-terminal domain of its largest subunit (CTD), will be continued. The CTD sequence requirements for cleavage in vitro will be determined. The role of the CTD in assembly of processing complexes will be investigated, including determination of which factor(s) directly contact(s) the cleavage site. Our recent discovery that CTD can bind directly to RNA will be pursued. A genetic system to study CTD function in vertebrate cells will be developed, and employed to examine the physiological significance and in vivo requirements of the CTD-RNA processing link. 3. Polyadenylation and transcription termination. Our recent findings that the transcriptional coactivator PC4/Sub1p interacts with polyadenylation factor CstF-64/Rna15p to function as an antiterminator will be extended. Biochemical and genetic experiments in yeast will be performed to investigate the association of these two factors withe elongating RNAP II, as well as the possible function of both CTD phosphorylation and capping enzymes in termination. Preliminary results suggesting that recombinant human capping enzyme can induce synthesis of a specific termination-related RNA by a phosphorylated CTD-T7 RNAP fusion protein will be extended. Minimal reconstituted systems employing CTD-T7 RNAP and purified capping and polyadenylation factors will be developed. 4. Polyadenylation and DNA repair. The recently described link between polyadenylation and DNA repair, mediated by an interaction between CstF and BARD1, will be further explored. Preliminary results suggesting that proteasomal degradation degradation of the RNAP II large subunit (LS) is involved in inhibition of 3' processing, will be confirmed and extended, including examination of UV-induced inhibition of processing in extracts of Cockayne's syndrome cells. Possible changes in the intranuclear distribution and colocalization of CstF, BARD1/BRCA1 and RNAP II will be. Cells lacking BARD1 will be obtained and used to examine the in vivo relevance and significance of the BARD1-mediated, DNA damage-induced in vitro inhibition of 3' cleavage. Finally, the possibility that the CstF-BARD1 interaction is functionally important for transcription-coupled repair will be investigated using DT40 cells conditionally expressing CstF-64
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