Dicer is an essential enzyme in all animals, where it cleaves double-stranded RNA (dsRNA) precursors to generate microRNAs (miRNAs) and small interfering RNAs (siRNAs). These small RNAs bind to messenger RNAs to regulate their expression. The proposed studies are focused on Dicer's helicase domain, which is most similar to a family of helicases involved in the innate immune response to viral infection. RIG-I, the founding member of this family, and Dicer, both recognize dsRNA termini. The goal of the proposed research is to understand how Dicer's helicase domain coordinates recognition of its dsRNA substrates, their termini and base-paired structures, to promote conformational changes that correlate with suboptimal (distributive) or optimal (processive) cleavage. Established biochemical assays will be used to perform a comprehensive analysis of Dicer's intrinsic substrate preferences. Features suggested by natural siRNA precursors, such as 5' and 3' overhangs of various lengths, and disruptions to base-pairing such as mismatches and loops, will be monitored for their ability to promote Dicer activities (binding, cleavage, and ATP hydrolysis), and to categorize substrates as optimal or suboptimal. dsRNA substrates that cannot be cleaved by Dicer alone will be assayed in the presence of dsRNA binding proteins (dsRBPs) that facilitate Dicer cleavage. Experiments to reveal the mechanism by which dsRBPs allow termini-independent cleavage will be conducted. The ability to define conditions that lead to distinct conformational states sets the stage for proposed cryo-electron microscopy and x-ray crystallography experiments. These are designed to obtain high- resolution information about the specific substrate interactions involved in the suboptimal and optimal conformations. Studies are designed to fill gaps in knowledge about how Dicer recognizes its naturally occurring substrates, both endogenous dsRNA and exogenous viral dsRNA. The proposed research will create new paradigms for researchers studying Dicer, and those seeking to manipulate Dicer activities for research or therapeutic uses.

Public Health Relevance

The proposed research focuses on Dicer, an enzyme essential for life. Studies are designed to reveal how Dicer recognizes its double-stranded RNA (dsRNA) substrates and distinguishes foreign dsRNA from naturally occurring dsRNA. The proposed studies set the stage for informed control of the immune response and manipulation of the enzyme for therapeutic and diagnostic purposes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM121706-02
Application #
9403224
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
2017-01-01
Project End
2020-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
2
Fiscal Year
2018
Total Cost
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
Sinha, Niladri K; Bass, Brenda L (2017) Overexpression and purification of Dicer and accessory proteins for biochemical and structural studies. Methods 126:54-65
Trettin, Kyle D; Sinha, Niladri K; Eckert, Debra M et al. (2017) Loquacious-PD facilitates Drosophila Dicer-2 cleavage through interactions with the helicase domain and dsRNA. Proc Natl Acad Sci U S A 114:E7939-E7948