Most eukaryotic messenger RNA precursors (pre-mRNAs) must undergo extensive co- transcriptional processing in the nucleus before they can be exported to the cytoplasm and function as mRNAs. The processing events include 5'-end capping, splicing, and 3'- end polyadenylation. The 3'-end polyadenylation of pre-mRNAs occurs in two steps- endonucleolytic cleavage of the pre-mRNA at a specific site near its 3'-end and then the addition of the poly(A) tail. While this process may appear to be simple biochemically, it requires a large number of protein factors for its execution, including cleavage and polyadenylation specificity factor (CSPF), cleavage stimulation factor (CstF), cleavage factors I and II, and poly(A) polymerase (PAP). The CPSF complex contains five subunits, CPSF-30, CPSF-73, CPSF-100, CPSF-160 and Fip1, and the CstF complex contains three subunits, CstF-50, CstF-64, and CstF-77. The 3'-end processing machinery in yeast has similarity to that in mammals, although there are also significant differences. During the previous funding period, we employed a divide-and-conquer approach to examine the various proteins of the mammalian and yeast 3'-end processing machineries on their own, and we have also begun to study their sub-complexes. A major emphasis for the current funding period is the studies on such complexes, which will produce significantly more insight into the molecular mechanism of these machineries. We will use symplekin/Pta1 as a unifying theme for some of these studies, as this scafold protein wil lead us to many other proteins in these machineries. We have determined the crystal structure of symplekin N-terminal domain in a ternary complex with the RNA polymerase II C-terminal domain (CTD) Ser5 phosphatase Ssu72 and a CTD phosphopeptide, illustrating a successful start for research in the current funding period.
Showing the most recent 10 out of 19 publications
