The experiments described in this proposal are designed to provide insights into the mechanism and regulation of pre-mRNA polyadenylation. Studies are proposed to understand how components of the polyadenylation machinery are regulated; how endonucleolytic cleavage is catalyzed; and how 3' end processing is linked to transcription. The following Specific Aims are proposed. 1. Regulation of polyadenylation. The mechanism and functional significance of the tissue-specific sumoylation of poly(A) polymerase (PAP) will be investigated. Whether sumoylation is required for PAP's essential function during cell growth will be determined, as will its effects on PAP stability, subcellular localization and enzymatic activity. In vitro assays will be used to investigate the mechanism of PAP sumoylation and to identify factor(s) responsible for tissue specificity. Symplekin is also modified by sumoylation, and this will be investigated using similar methods. Findings that cells expressing a phosphorylation-defective PAP specifically overexpress c-jun mRNA with lengthened poly(A) tails will be explored. Tumor-derived cell lines that overexpress PAP will be analyzed to determine whether c-jun (and/or c-fos) poly(A) tails are lengthened. 2. Mechanism and control of 3' cleavage. Studies indicating that CPSF-73 is the endonuclease responsible for 3' cleavage will be pursued. Recent findings that CPSF-73 displays nuclease activity in vitro will be confirmed and extended. Key residues in the p-lactamase domain will be mutated, and effects on catalysis determined. Nuclease activity will be characterized in several ways, including analysis of the role of the newly described CASP domain. The role of CPSF-100, which contains a domain structure very similar to that of CPSF-73 but lacks catalytic activity, will be studied. The possibility that sumoylation regulates CPSF-73/100 will be investigated. The potential role in polyadenylation of RC-68 and RC-74, which share similarity with CPSF-73 and CPSF-100, respectively, will be explored. 3. Transcription and 3' processing. Recent findings that the RNA polymerase II CTD binds RNA selectively and that this can suppress transcription-coupled 3' end formation will be pursued. Cellular genes that are possible targets will be examined by mutation of suspected sequences and by determining whether the CTD interacts with these sequences. The mechanism of 3' processing inhibition will be investigated in vitro, and the role of specific CTD heptad repeats studied using genetic analysis in DT40 cells. Sub1/PC4 functions to help connect transcription and 3' processing, and data suggesting that this reflects interactions with CTD modifying enzymes will be pursued. Whether these functions are conserved in humans will be determined. The function of the PAF complex, known in yeast to play a role in linking transcription with CTD and chromatin modification, and perhaps 3' end formation, will be analyzed in human cells. RNAi will be used to deplete PAF components in vivo and the effects on 3' end formation will be determined. Finally, a reconstituted coupled transcription-3' processing system will be developed and, using chromatinized templates, the function of PAF in linking transcription, chromatin modification and 3' processing analyzed. ? ? ?
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