The subject of this research project is the structural molecular biology of RNA processing and of the coupling with transcription. Although RNA processing reactions can be reconstituted in vitro, they occur more efficiently in the cell because they are closely integrated with transcription. How these processes are regulated and integrated with each other remains unclear at the molecular level and even less well understood in structural terms. The specific goal of this proposal is to study how the formation of the mature 3'-end of a messenger RNA by cleavage and polyadenylation is coupled to the termination of transcription and to the subsequent export of the mature RNA from the cell nucleus. In order to address these biological problems, we propose to study: (1) the molecular basis for RNA recognition by RNA processing factors Rna15 and Hrp1, and RNA export factor Npl3, to understand how RNA-binding proteins within the 3'-end processing complex are organized on the processing signals;(2) the interaction of Rna15 with the transcription termination factor Pcf11, to understand how the chain of events leading to transcription termination is initiated once a polydenylation site is transcribed;(3) the molecular basis for the specific recognition of different phosphorylatyed forms of the C- terminal domain of the RNA polymerase by transcription termination factors Rtt103 and Pcf11;(4) how the poly(A) polymerase enzyme responsible for addition of the poly(A) tail is recruited by Fip1 so that the enzyme is only active on pre-mRNAs;(5) the structure of the heterotrimeric protein complex (CstF) that recognizes the 3'-end processing sites of vertebrate mRNAs and couples RNA processing with transcription termination. The integration of gene expression mechanisms adds robustness and allows for multiple levels of regulation. Therefore, dissecting how transcription and RNA processing are coupled at the molecular and structural level, as we propose to do, is an essential step to understanding how gene expression pathways are integrated and regulated.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM064440-08
Application #
7799865
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
2002-07-01
Project End
2012-05-31
Budget Start
2010-04-01
Budget End
2012-05-31
Support Year
8
Fiscal Year
2010
Total Cost
$371,576
Indirect Cost
Name
University of Washington
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Yang, Fan; Hsu, Peter; Lee, Susan D et al. (2017) The C terminus of Pcf11 forms a novel zinc-finger structure that plays an essential role in mRNA 3'-end processing. RNA 23:98-107
Nemec, Corey M; Yang, Fan; Gilmore, Joshua M et al. (2017) Different phosphoisoforms of RNA polymerase II engage the Rtt103 termination factor in a structurally analogous manner. Proc Natl Acad Sci U S A 114:E3944-E3953
Borkar, Aditi N; Bardaro Jr, Michael F; Camilloni, Carlo et al. (2016) Structure of a low-population binding intermediate in protein-RNA recognition. Proc Natl Acad Sci U S A 113:7171-6
Hopping, Gene; Kellock, Jackson; Barnwal, Ravi Pratap et al. (2014) Designed ?-sheet peptides inhibit amyloid formation by targeting toxic oligomers. Elife 3:e01681
Hsu, Peter L; Yang, Fan; Smith-Kinnaman, Whitney et al. (2014) Rtr1 is a dual specificity phosphatase that dephosphorylates Tyr1 and Ser5 on the RNA polymerase II CTD. J Mol Biol 426:2970-81
Smith-Kinnaman, Whitney R; Berna, Michael J; Hunter, Gerald O et al. (2014) The interactome of the atypical phosphatase Rtr1 in Saccharomyces cerevisiae. Mol Biosyst 10:1730-41
Sperber, Henrik; Beem, Alan; Shannon, Sandra et al. (2014) miRNA sensitivity to Drosha levels correlates with pre-miRNA secondary structure. RNA 20:621-31
Chen, Yu; Varani, Gabriele (2013) Engineering RNA-binding proteins for biology. FEBS J 280:3734-54
Barnwal, Ravi Pratap; Lee, Susan D; Moore, Claire et al. (2012) Structural and biochemical analysis of the assembly and function of the yeast pre-mRNA 3' end processing complex CF I. Proc Natl Acad Sci U S A 109:21342-7
Walbott, Hélène; Machado-Pinilla, Rosario; Liger, Dominique et al. (2011) The H/ACA RNP assembly factor SHQ1 functions as an RNA mimic. Genes Dev 25:2398-408

Showing the most recent 10 out of 20 publications