The experiments described in this proposal are designed to provide insight into the mechanism and regulation of pre-mRNA polyadenylation. The following Specific Aims are proposed. 1. Structural studies on 3'processing. Studies with Liang Tong to determine the x-ray structures of a CPSF-73/CPSF-100 complex and of the scaffolding protein symplekin will be pursued. Studies on the former will be complemented with endonuclease assays employing the purified complex as well as structure-based mutant derivatives of CPSF- 73. Recent structural studies of the symplekin N terminus will be pursued, and efforts to crystallize the evolutionarily conserved central domain continued. Using previously developed methods to purify the assembled 3'processing complex, a collaboration with Joachim Frank will be continued, with the goal of obtaining a 3D reconstruction of the entire complex by cryo EM. 2. 3'processing and links to transcription. Experiments examining the function of the multisubunit PAF complex in linking transcription and processing using an in vitro coupled reaction will be pursued. Our recent finding that a model transcription factor, GAL4- VP16, can directly activate 3'processing, will be further investigated. Genetic studies employing DT40 cells expressing a conditional RNA polymerase II largest subunit (LS) will be pursued to investigate the role of the LS C-terminal domain (CTD) in 3'processing. Studies on transcription termination will include structure-based analysis of the yeast 5'3'exonuclease Rat1. The possible role of the human protein Senataxin, the apparent homologue of the yeast RNA-DNA helicase Sen1, in transcription-termination will be investigated. 3. 3'processing factors and their regulation. Two proteins discovered in our recent proteomic analysis of the 3'processing complex, Rbbp6 (Rb binding protein 6) and PARP1 (poly(ADP) ribosylase 1), will be investigated. Experiments suggesting that Rbbp6 is necessary for 3'cleavage will be pursued. The role of a RING domain and domains binding the tumor suppressors p53 and Rb will be analyzed. Data indicating that PARP1 regulates polyadenylation, perhaps by PARylating poly(A) polymerase (PAP), will be extended, and the effects of agents that induce PARP1 on polyadenylation determined. PAP is extensively modified by sumoylation in a tissue-specific manner. Experiments that identified an E3 SUMO ligase, PIAS3, that enhances PAP sumoylation will be extended, and the possibility that PIAS3 and/or the SUMO protease SENP1 are involved in tissue specificity explored. The function of the evolutionarily conserved sumoylation of CPSF-73 will be investigated by analysis of the yeast homologue, Ysh1. Finally, recent studies of others have indicated that global changes in alternative polyadenylation occur during cell differentiation. The mechanistic basis for this will be investigated with a model in vitro system using C2C12 myoblasts, which will allow identification of the factor(s) involved. 3'processing will be reconstituted with purified factors, which will allow analysis of how variations in concentrations of specific components influence poly(A) site choice.
The experiments described in this proposal are designed to increase our understanding of the mechanism and regulation of the essential reaction that creates the polyadenylated 3'ends of mRNA. Recent studies have revealed that changes in polyadenylation occur during development and disease, and our studies will provide a mechanistic understanding for these changes.
|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|
|Di Giammartino, Dafne Campigli; Li, Wencheng; Ogami, Koichi et al. (2014) RBBP6 isoforms regulate the human polyadenylation machinery and modulate expression of mRNAs with AU-rich 3' UTRs. Genes Dev 28:2248-60|
|Chan, Serena L; Huppertz, Ina; Yao, Chengguo et al. (2014) CPSF30 and Wdr33 directly bind to AAUAAA in mammalian mRNA 3' processing. Genes Dev 28:2370-80|
|Di Giammartino, Dafne Campigli; Manley, James L (2014) New links between mRNA polyadenylation and diverse nuclear pathways. Mol Cells 37:644-9|
|Morales, Julio C; Richard, Patricia; Rommel, Amy et al. (2014) Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair. Nucleic Acids Res 42:4996-5006|
|Nagaike, Takashi; Manley, James L (2014) In vitro analysis of transcriptional activators and polyadenylation. Methods Mol Biol 1125:65-74|
|Richard, Patricia; Feng, Shuang; Manley, James L (2013) A SUMO-dependent interaction between Senataxin and the exosome, disrupted in the neurodegenerative disease AOA2, targets the exosome to sites of transcription-induced DNA damage. Genes Dev 27:2227-32|
|Vi, Son Lang; Trost, Gerda; Lange, Peggy et al. (2013) Target specificity among canonical nuclear poly(A) polymerases in plants modulates organ growth and pathogen response. Proc Natl Acad Sci U S A 110:13994-9|
|Richard, Patricia; Manley, James L (2013) How bidirectional becomes unidirectional. Nat Struct Mol Biol 20:1022-4|
|Di Giammartino, Dafne Campigli; Shi, Yongsheng; Manley, James L (2013) PARP1 represses PAP and inhibits polyadenylation during heat shock. Mol Cell 49:7-17|
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