The human body contains hundreds of thousands of unique small regulatory RNAs that play a substantial role in shaping human gene expression. The majority of these small RNAs can be classified into one of three types: microRNAs (miRNA), small interfering RNAs (siRNA) and piwi-interacting RNAs (piRNA). All three types of small RNA function as guides for members of the Argonaute protein family, which use the encoded sequence information to identify genes and transcripts targeted for silencing. Therefore, the structure and dynamics of the Argonaute proteins dictate the base pairing requirements for target recognition by small RNAs. Importantly, Argonautes do not necessarily require perfect complementarity to their small RNA guide to recognize a target, and the three types of small RNAs are used by the Argonautes in divergent ways. Consequently, understanding and predicting how small RNAs find their targets remains a substantial outstanding challenge with broad implications for understanding regulation of human gene expression. The objective of the proposed research is to provide fundamental insight into the unique features underlying target recognition by the three small RNAs families. Our approach is to leverage Argonaute structure to inform biochemical experiments aimed at identifying modes of target recognition.
In Aim 1 we propose to establish criteria for identifying non-conical targeting by miRNAs, which is currently difficult to predict and yet makes up a substantial portion of miRNA:target interactions in cells. Results are expected to enable miRNA target prediction and thereby facilitate researchers studying diverse aspects of human gene regulation.
In Aim 2 we will define the conformational switch that Argonaute undergoes in order to transition from using miRNA to siRNA silencing mechanisms. Results are expected to inform siRNA design and enable those harnessing siRNAs to treat patients.
In Aim 3 we will determine rules for target recognition by piRNAs, the largest and most poorly understood class of human small RNAs. Expected results will provide fundamental knowledge necessary for deciphering the role of piRNAs in regulation of human gene expression.

Public Health Relevance

RNA silencing is a natural process by which small RNAs ?silence? genes (or turn genes off), and is required for normal life span, brain development, and prevention of cancer. This project aims to understand the three major classes of small RNAs in humans. The proposed research will lead to a better understanding of human gene regulation and contribute to on-going efforts to harness RNA silencing as a new type of therapeutic for the treatment of human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM104475-06
Application #
9552180
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Bender, Michael T
Project Start
2013-08-01
Project End
2019-06-30
Budget Start
2018-07-01
Budget End
2019-06-30
Support Year
6
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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Ziv, Omer; Gabryelska, Marta M; Lun, Aaron T L et al. (2018) COMRADES determines in vivo RNA structures and interactions. Nat Methods 15:785-788
Klum, Shannon M; Chandradoss, Stanley D; Schirle, Nicole T et al. (2018) Helix-7 in Argonaute2 shapes the microRNA seed region for rapid target recognition. EMBO J 37:75-88
Sheu-Gruttadauria, Jessica; MacRae, Ian J (2017) Structural Foundations of RNA Silencing by Argonaute. J Mol Biol 429:2619-2639
Suter, Scott R; Sheu-Gruttadauria, Jessica; Schirle, Nicole T et al. (2016) Structure-Guided Control of siRNA Off-Target Effects. J Am Chem Soc 138:8667-9
Schirle, Nicole T; Kinberger, Garth A; Murray, Heather F et al. (2016) Structural Analysis of Human Argonaute-2 Bound to a Modified siRNA Guide. J Am Chem Soc 138:8694-7
Schirle, Nicole T; Sheu-Gruttadauria, Jessica; Chandradoss, Stanley D et al. (2015) Water-mediated recognition of t1-adenosine anchors Argonaute2 to microRNA targets. Elife 4:
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