The long-term goal of this proposal is to understand, in detail, the mechanisms of mammalian mRNA 3'processing through comprehensive characterization of the 3'processing complex. mRNA 3'-end formation is an essential step of gene expression in eukaryotes, and has profound influences on many aspects of RNA metabolism, including mRNA stability, export, and translation. Additionally, regulated mRNA 3'processing has emerged as a critical mechanism for gene control. Aberrant 3'processing signals and mutant 3'processing factors cause a number of human diseases, including cancer. For almost all eukaryotic mRNAs, 3'processing involves two catalytic steps, an endonucleolytic cleavage followed by the addition of a poly(A) tail. Both of these steps take place in a macromolecular machinery, the 3'processing complex. The structure-function relationship of this complex is a central question in understanding the mechanism of mRNA 3'processing. We have recently purified the functional 3'processing complex assembled on its mRNA target and initiated proteomic, functional, and structural analyses. Building on our purification system, we will address several fundamental and long-standing questions in the field: How are the 3'processing signals in mRNAs specifically recognized? What is the protein composition of the 3'processing complex and (how) does it change during 3'processing reaction? What is the structure of the 3'processing complex? To achieve our objectives, we have designed the following specific aims: 1. Comprehensively map the protein-RNA interactions within the purified 3'processing complex. 2. Characterize the compositional changes of the 3'processing complex during 3'processing reaction. 3. Characterize the structural changes of the 3'processing complex during 3'processing reaction using single particle cryo-electron microscopy (cryo-EM). Accomplishment of the proposed studies will provide new and significant mechanistic insights into the structure-function relationship and the dynamics of the mRNA 3'processing complex, and may also provide the foundation for development of new therapeutic approaches against diseases caused by aberrant 3'processing.
mRNA 3'-end formation is an essential step of eukaryotic gene expression. It plays an important role in gene regulation, and aberrant 3'-end formation has been implicated in a variety of human diseases. The goal of this project is to understand the mechanism of mRNA 3'processing through comprehensive characterization of the 3'processing machinery.
|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|
|Lackford, Brad; Yao, Chengguo; Charles, Georgette M et al. (2014) Fip1 regulates mRNA alternative polyadenylation to promote stem cell self-renewal. EMBO J 33:878-89|
|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|
Showing the most recent 10 out of 20 publications