When double-stranded RNA (dsRNA) is introduced into a cell, the cognate mRNA is degraded and its gene is silenced. This phenomenon is called RNA interference (RNAi) and occurs through a recently discovered biological pathway. An early step in RNAi is cleavage of the dsRNA to small pieces called siRNAs. Members of the RNase III family of enzymes are thought to catalyze this reaction, and the goal of this proposal is to understand how these enzymes participate in the process. This goal will be approached by characterizing the intrinsic biochemical properties of RNase III family members, and correlating these data with observations about RNAi made in vivo using Caenorhabditis elegans. Comparisons between wild-type animals, and animals containing deletions in each of the two C. elegans RNase III genes, will be used to correlate in vivo and in vitro data. GFP reporter constructs and immunofluorescence will be used to determine the expression patterns of the enzymes. Mutant animals, extracts prepared from these animals, and purified enzymes, will be assayed for their ability to produce siRNAs. Nucleic acid binding properties of the enzymes, and the structure of their RNA cleavage products, will be used to distinguish between mechanisms suggested by a recent crystal structure of RNase III and existing information about RNAi. Phenotypes of C. elegans strains lacking the RNase III enzymes will be characterized with an aim towards defining the roles of the enzymes in nuclear pathways. Microarray technology will be used to search for natural dsRNA substrates and their targets, and genes identified in a recent screen will be tested for nuclear functions in gene silencing. These studies will lead to information that will be helpful in using RNAi as a therapeutic agent, and also offer further insight into this recently discovered biological pathway. Understanding this biological pathway is crucial to understanding how it intersects with medically relevant pathways.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067106-04
Application #
7009985
Study Section
Special Emphasis Panel (ZRG1-CDF-1 (01))
Program Officer
Rhoades, Marcus M
Project Start
2003-02-01
Project End
2009-01-31
Budget Start
2006-02-01
Budget End
2009-01-31
Support Year
4
Fiscal Year
2006
Total Cost
$153,287
Indirect Cost
Name
University of Utah
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Welker, Noah C; Maity, Tuhin S; Ye, Xuecheng et al. (2011) Dicer's helicase domain discriminates dsRNA termini to promote an altered reaction mode. Mol Cell 41:589-99
Warf, M Bryan; Johnson, W Evan; Bass, Brenda L (2011) Improved annotation of C. elegans microRNAs by deep sequencing reveals structures associated with processing by Drosha and Dicer. RNA 17:563-77
Evan Johnson, W; Welker, Noah C; Bass, Brenda L (2011) Dynamic linear model for the identification of miRNAs in next-generation sequencing data. Biometrics 67:1206-14
Aruscavage, P Joseph; Hellwig, Sabine; Bass, Brenda L (2010) Small DNA pieces in C. elegans are intermediates of DNA fragmentation during apoptosis. PLoS One 5:e11217
Welker, Noah C; Pavelec, Derek M; Nix, David A et al. (2010) Dicer's helicase domain is required for accumulation of some, but not all, C. elegans endogenous siRNAs. RNA 16:893-903
Habig, Jeffrey W; Aruscavage, P Joseph; Bass, Brenda L (2008) In C. elegans, high levels of dsRNA allow RNAi in the absence of RDE-4. PLoS One 3:e4052
Parker, Greg S; Maity, Tuhin Subhra; Bass, Brenda L (2008) dsRNA binding properties of RDE-4 and TRBP reflect their distinct roles in RNAi. J Mol Biol 384:967-79
Habig, Jeffrey W; Dale, Taraka; Bass, Brenda L (2007) miRNA editing--we should have inosine this coming. Mol Cell 25:792-3
Welker, Noah C; Habig, Jeffrey W; Bass, Brenda L (2007) Genes misregulated in C. elegans deficient in Dicer, RDE-4, or RDE-1 are enriched for innate immunity genes. RNA 13:1090-102
Bass, B L (2006) How does RNA editing affect dsRNA-mediated gene silencing? Cold Spring Harb Symp Quant Biol 71:285-92

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