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.

Public Health Relevance

Project Narrative Defects in pre-mRNA processing are linked to human diseases. Our studies will contribute to understanding the molecular mechanism for pre-mRNA 3'-end processing.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM077175-05A1
Application #
8234525
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Preusch, Peter C
Project Start
2007-07-01
Project End
2016-02-29
Budget Start
2012-04-01
Budget End
2013-02-28
Support Year
5
Fiscal Year
2012
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
Xiang, Kehui; Tong, Liang; Manley, James L (2014) Delineating the structural blueprint of the pre-mRNA 3'-end processing machinery. Mol Cell Biol 34:1894-910
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
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
Wilson, William C; Hornig-Do, Hue-Tran; Bruni, Francesco et al. (2014) A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression. Hum Mol Genet 23:6345-55
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
Zhang, Jun; Tan, Dazhi; DeRose, Eugene F et al. (2014) Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation. Proc Natl Acad Sci U S A 111:E2937-46
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
Paulson, Ashley R; Tong, Liang (2012) Crystal structure of the Rna14-Rna15 complex. RNA 18:1154-62
Bai, Yun; Srivastava, Sandeep K; Chang, Jeong Ho et al. (2011) Structural basis for dimerization and activity of human PAPD1, a noncanonical poly(A) polymerase. Mol Cell 41:311-20
Chang, Jeong Ho; Xiang, Song; Xiang, Kehui et al. (2011) Structural and biochemical studies of the 5'?3' exoribonuclease Xrn1. Nat Struct Mol Biol 18:270-6

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