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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM077175-08
Application #
8811968
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Preusch, Peter
Project Start
2007-07-01
Project End
2016-02-29
Budget Start
2015-03-01
Budget End
2016-02-29
Support Year
8
Fiscal Year
2015
Total Cost
$384,872
Indirect Cost
$139,336
Name
Columbia University (N.Y.)
Department
Biology
Type
Other Domestic Higher Education
DUNS #
049179401
City
New York
State
NY
Country
United States
Zip Code
10027
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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Luo, Shukun; Tong, Liang (2014) Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain. Proc Natl Acad Sci U S A 111:13834-9
Jurado, Ashley R; Tan, Dazhi; Jiao, Xinfu et al. (2014) Structure and function of pre-mRNA 5'-end capping quality control and 3'-end processing. Biochemistry 53:1882-98
Tan, Dazhi; Zhou, Mi; Kiledjian, Megerditch et al. (2014) The ROQ domain of Roquin recognizes mRNA constitutive-decay element and double-stranded RNA. Nat Struct Mol Biol 21:679-85
Tan, Dazhi; Marzluff, William F; Dominski, Zbigniew et al. (2013) Structure of histone mRNA stem-loop, human stem-loop binding protein, and 3'hExo ternary complex. Science 339:318-21
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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