The long-term goal of this proposal is to understand, in detail, the mechanisms of mammalian mRNA 3' processing and its regulation. mRNA 3'-end formation, typically involving an endonucleolytic cleavage followed by polyadenylation, is an essential step of eukaryotic gene expression and it significantly impacts many aspects of RNA metabolism, including mRNA stability and translation. In addition, the majority of eukaryotic genes produce multiple mRNA isoforms with distinct 3' ends through alternative polyadenylation (APA). Recent studies have revealed that APA is highly regulated in development and plays an important role in post-transcriptional gene regulation. Aberrant APA patterns have been associated with a wide range of diseases, from cancer to neuromuscular disorders. As such, a central question in the mRNA 3' processing field has been how polyadenylation sites (PAS) are recognized and how PAS selection can be regulated. The majority of mammalian PAS contain an AAUAAA hexamer and a U/UG-rich downstream element (DSE). According to the current model in the field, these key cis-elements are specifically recognized by CPSF160 of the CPSF complex and CstF64 of the CstF complex respectively. However, our published and preliminary data have challenged this model in several key aspects: 1) we have recently shown that the CPSF subunits CPSF30 and Wdr33, but not CPSF160 as was generally believed, directly bind to AAUAAA; 2) we have provided evidence that maintenance of the CPSF-RNA interaction specificity requires a novel proofreading factor(s) (see preliminary data); 3) we have demonstrated that the general 3' processing factor CstF64 in fact only binds to a subset of mammalian PAS, and we provided evidence that recognition of PAS with different sequence features requires distinct RNA-binding protein(s) (see preliminary data). These observations have not only significantly changed the current model for PAS recognition, but also revealed much greater complexity in mRNA 3' processing than previously appreciated. Here we propose to better define the molecular mechanisms of mammalian PAS recognition through an in-depth characterization of the key protein-RNA interactions involved. Accomplishing the proposed research will provide seminal insights into the fundamental mechanisms of mammalian mRNA 3' processing and its regulation. As aberrant PAS selection has been associated with a broad spectrum of human diseases, a better understanding of the mechanisms for PAS recognition may provide the foundation for the development of new therapeutics for these diseases.

Public Health Relevance

mRNA 3' processing is not only an essential step in eukaryotic gene expression, it also plays important roles in gene regulation. Aberrant mRNA 3' processing causes a wide variety of human diseases. The goal of this project is to understand the mechanisms and regulation of mRNA 3' processing by an in- depth characterization of the protein-RNA interactions in the mRNA 3' processing complex.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM090056-07
Application #
9130928
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
2010-03-15
Project End
2019-04-30
Budget Start
2016-05-01
Budget End
2017-04-30
Support Year
7
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of California Irvine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92617
Zhu, Yong; Wang, Xiuye; Forouzmand, Elmira et al. (2018) Molecular Mechanisms for CFIm-Mediated Regulation of mRNA Alternative Polyadenylation. Mol Cell 69:62-74.e4
Sun, Yadong; Zhang, Yixiao; Hamilton, Keith et al. (2018) Molecular basis for the recognition of the human AAUAAA polyadenylation signal. Proc Natl Acad Sci U S A 115:E1419-E1428
Brumbaugh, Justin; Di Stefano, Bruno; Wang, Xiuye et al. (2018) Nudt21 Controls Cell Fate by Connecting Alternative Polyadenylation to Chromatin Signaling. Cell 172:106-120.e21
Huang, Chunliu; Shi, Junjie; Guo, Yibin et al. (2017) A snoRNA modulates mRNA 3' end processing and regulates the expression of a subset of mRNAs. Nucleic Acids Res 45:8647-8660
Movassat, Maliheh; Crabb, Tara L; Busch, Anke et al. (2016) Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns. RNA Biol 13:646-55
Weng, Lingjie; Li, Yi; Xie, Xiaohui et al. (2016) Poly(A) code analyses reveal key determinants for tissue-specific mRNA alternative polyadenylation. RNA 22:813-21
Zou, Donghua; McSweeney, Colleen; Sebastian, Aswathy et al. (2015) A critical role of RBM8a in proliferation and differentiation of embryonic neural progenitors. Neural Dev 10:18
Shi, Yongsheng; Manley, James L (2015) The end of the message: multiple protein-RNA interactions define the mRNA polyadenylation site. Genes Dev 29:889-97
Yao, Chengguo; Weng, Lingjie; Shi, Yongsheng (2014) Global protein-RNA interaction mapping at single nucleotide resolution by iCLIP-seq. Methods Mol Biol 1126:399-410
Yao, Chengguo; Shi, Yongsheng (2014) Global and quantitative profiling of polyadenylated RNAs using PAS-seq. Methods Mol Biol 1125:179-85

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