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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics A Study Section (MGA)
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Preusch, Peter C
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Columbia University (N.Y.)
Other Domestic Higher Education
New York
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Zhang, Yinglu; Rataj, Katarzyna; Simpson, Gordon G et al. (2016) Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA. PLoS One 11:e0160694
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Xiang, Kehui; Manley, James L; Tong, Liang (2012) An unexpected binding mode for a Pol II CTD peptide phosphorylated at Ser7 in the active site of the CTD phosphatase Ssu72. Genes Dev 26:2265-70
Paulson, Ashley R; Tong, Liang (2012) Crystal structure of the Rna14-Rna15 complex. RNA 18:1154-62

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