Short interfering RNAs (siRNAs) are small RNA regulators capable of reducing protein expression of their target mRNAs in a process known as RNA interference (RNAi). RNAi is important for normal cellular physiology and adaptation, as well as an invaluable genetic tool for scientists and a promising source of clinical therapeutics. While canonical models of RNAi feature siRNA-directed target mRNA cleavage by Argonaute proteins (Agos), recent studies have demonstrated that Ago-mediated cleavage is dispensable for target mRNA repression. Additional studies and comparisons with micro RNAs suggest that siRNAs may repress their target mRNAs via Ago-dependent translational repression and/or stimulation of degradation through non-Ago nucleases. To determine the mechanism(s) of siRNA-mediated mRNA repression, I propose a two-pronged approach: (1) a thorough analysis of siRNA-dependent changes in translation and degradation for siRNA target mRNAs in vivo and (2) biochemical purification and characterization of Ago-siRNA complexes and their interacting protein partners. I will utilize high throughput sequencing technologies to test severa different models of siRNA-dependent effects on translation and degradation in both C. elegans and H. sapiens, and take advantage of the wealth of functional genetic information in C. elegans to determine the cellular machinery required for any observed effect(s). To complement this approach I will directly identify the complexes associated with mRNA repression and assess their contributions to mRNA repression, again exploiting C. elegans' genetic resources. These approaches will provide a more detailed and comprehensive view of siRNA- dependent mechanism(s) of mRNA repression, and the techniques employed will be applicable to siRNA studies in many systems. Ultimately, knowledge of siRNAs' molecular effectors and mechanism(s) will be necessary for harnessing RNAi's full clinical and research potential, understanding the biological basis of off- target effects, and furthering our understanding of small RNA biology.

Public Health Relevance

The versatility of RNA interference (RNAi) for silencing gene expression has led to its widespread adoption by researchers, and has made it a promising source of clinical therapeutics. However, current approaches toward applying RNAi have been limited by a variety of factors, many of which stem from a lack of fundamental knowledge on the metabolism, reactivities, and functions of small RNA effectors in animal systems. Knowledge of RNAi's mechanism(s) will be useful for RNAi to realize its full clinical and research potential, with relevance to our understanding of the underlying biology of RNAi-based gene regulation, the design of more effectual RNAi treatments, why RNAi at times fails, and the biological basis of off-target effects.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM112474-03
Application #
9187825
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Hoodbhoy, Tanya
Project Start
2014-12-01
Project End
2017-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
3
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Stanford University
Department
Pathology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
Botham, Crystal M; Arribere, Joshua A; Brubaker, Sky W et al. (2017) Ten simple rules for writing a career development award proposal. PLoS Comput Biol 13:e1005863
Arribere, Joshua A; Cenik, Elif S; Jain, Nimit et al. (2016) Translation readthrough mitigation. Nature 534:719-23
Saha, Agniva; Mitchell, Jessica A; Nishida, Yuri et al. (2015) A trans-dominant form of Gag restricts Ty1 retrotransposition and mediates copy number control. J Virol 89:3922-38
Arribere, Joshua A; Bell, Ryan T; Fu, Becky X H et al. (2014) Efficient marker-free recovery of custom genetic modifications with CRISPR/Cas9 in Caenorhabditis elegans. Genetics 198:837-46