RNA interference (RNAi) and related pathways trigger potent and specific regulation of gene expression in eukaryotes. Gene silencing begins with the targeting of messenger RNAs complementary to 21-nucleotide guide RNAs called short interfering RNAs (siRNAs) or microRNAs (miRNAs), ultimately leading to destruction of the targeted transcript. Two specialized cytoplasmic ribonucleases function at either end of this pathway: the Dicer endonuclease catalyzes length-specific double-strand RNA cleavage to produce si- and miRNAs, whereas the Xrn1 exonuclease degrades targeted mRNAs after initial cleavage by the RNA-induced silencing complex (RISC). The central objective of this project is to determine the structural basis for RNA recognition and processing by Dicer and Xrn1 enzymes, and to elucidate the respective functions and interactions of these enzymes with other proteins. The investigation of Dicer builds on our success during the past project period in determining Dicer structures and biochemical activities. The study of Xrn1 represents an important addition to the overall project, aimed at dissecting the mechanism by which targeted mRNAs are destroyed. Focusing on the human and Drosophila Dicer and Xrn1 enzymes, our specific aims are to determine how dicing activity is regulated, to test the roles of double-stranded RNA binding proteins in Dicer substrate specificity and guide strand selection, and to solve and analyze molecular structures of Xrn1. The significance of RNAi as an essential and widespread mechanism of gene regulation in eukaryotes underscores the need for a fundamental understanding of these enzymes. Knowledge of the molecular mechanisms that govern RNAi will enable the engineering of these activities for therapeutic purposes.

Public Health Relevance

RNA interference (RNAi) and related pathways involve small RNA molecules that bind to messenger RNAs to alter the levels of proteins produced in cells. Our discoveries about how such regulatory RNAs are produced and utilized in human cells will enable academic and industrial laboratories to harness RNAi to enhance or repress production of specific proteins for therapeutic purposes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM073794-07
Application #
8245806
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
2005-09-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
7
Fiscal Year
2012
Total Cost
$285,119
Indirect Cost
$95,419
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Sternberg, Samuel H; Redding, Sy; Jinek, Martin et al. (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507:62-7
Kidwell, Mary Anne; Chan, Jessica M; Doudna, Jennifer A (2014) Evolutionarily conserved roles of the dicer helicase domain in regulating RNA interference processing. J Biol Chem 289:28352-62
Taylor, David W; Ma, Enbo; Shigematsu, Hideki et al. (2013) Substrate-specific structural rearrangements of human Dicer. Nat Struct Mol Biol 20:662-70
Lee, Ho Young; Zhou, Kaihong; Smith, Alison Marie et al. (2013) Differential roles of human Dicer-binding proteins TRBP and PACT in small RNA processing. Nucleic Acids Res 41:6568-76
Koh, Hye Ran; Kidwell, Mary Anne; Ragunathan, Kaushik et al. (2013) ATP-independent diffusion of double-stranded RNA binding proteins. Proc Natl Acad Sci U S A 110:151-6
Pattanayak, Vikram; Lin, Steven; Guilinger, John P et al. (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31:839-43
Noland, Cameron L; Ma, Enbo; Doudna, Jennifer A (2011) siRNA repositioning for guide strand selection by human Dicer complexes. Mol Cell 43:110-21
Jinek, Martin; Coyle, Scott M; Doudna, Jennifer A (2011) Coupled 5' nucleotide recognition and processivity in Xrn1-mediated mRNA decay. Mol Cell 41:600-8
Chakravarthy, Srinivas; Sternberg, Samuel H; Kellenberger, Colleen A et al. (2010) Substrate-specific kinetics of Dicer-catalyzed RNA processing. J Mol Biol 404:392-402
Jinek, Martin; Fabian, Marc R; Coyle, Scott M et al. (2010) Structural insights into the human GW182-PABC interaction in microRNA-mediated deadenylation. Nat Struct Mol Biol 17:238-40

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