The principal goal of this research project is to understand how pre-mRNA polyadenylation occurs. Work during the previous granting period has shown that this is an unexpectedly complex reaction, requiring approximately 10 distinct polypeptides. The factors involved include two multisubunit RNA binding complexes, CPSF and CstF, two cleavage factors, CFI and ll, and poly(A) polymerase (PAP). Both in vitro and in vivo assays will be employed to study the mechanism and regulation of polyadenylation. The following specific aims are proposed: 1. Purification and cloning of remaining polyadenylation factors. While a number of these have already been purified and/or cloned, several remain. Most notable are two partially purified proteins, cleavage factors I and ll, one or both of which almost certainly constitute the actual endonuclease that cleaves the pre-mRNA. These proteins will be purified to homogeneity, most likely from calf-thymus, and cDNAS cloned using protein microsequence information. 2. Biochemistry of polyadenylation. The multiple protein-RNA and protein- protein interactions required for polyadenylation will be investigated in detail. These include binding of CPSF, CstF and PAP to RNA; the protein- protein interactions responsible for the cooperative RNA binding of CPSF and CstF; interactions of PAP with CPSF; and the interaction of the cleavage factors with other components. 3. In vivo analysis of polyadenylation. Efforts will be made to develop in vivo systems to study polyadenylation. Several approaches will be employed to study interactions between polyadenylation factors in transiently transfected cells. Drosophila homologues of selected genes will be isolated and their chromosomal locations determined. Any genes that map to positions of previously described mutants that might conceivably play a role in RNA processing will be investigated further. A chicken B cell line that undergoes high levels of homologous recombination will be used to determine whether selected genes are required for viability. 4. Regulation of polyadenylation. This will be studied from two perspectives: First, changes in the expression of the 64 kD subunit of CstF during B cell development will be studied as a specific example. The possibility that such changes influence the alternative processing of IgM (membrane vs. secreted) pre-mRNA will be investigated. Second, possible regulation of poly(A) polymerase, and appropriate other factors, including 64kD, will be examined more generally. Distinctive features of their expression, such as the existence of alternatively spliced mRNAs, and structure (e.g., each are phosphoproteins) suggest that their activities may be regulated. Expression patterns and activities in various cell types will be investigated, and the significance of any differences examined.
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