Supplement: Defining and Controlling Protein-RNA interactions in editing and interference pathways

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This Maximizing Investigators Research Award (MIRA) application is proposed to support research in the Beal lab at UC Davis focused on defining and controlling protein-RNA interactions in RNA editing and RNA interference pathways. The RNA editing ADAR enzymes convert adenosines (A) to inosines (I) in duplex RNA. Since I can behave similarly to guanosine (G) in RNA, this modification can have profound effects on the structure and function of the modified RNA including, but not limited to, changes in the meaning of specific codons (recoding). Mutations in the human ADAR1 gene cause the skin disorder Dyschromatosis Symmetrica Hereditaria (DSH) and the autoimmune disease Aicardi- Goutieres Syndrome (AGS). Also, ADAR1 upregulation and hyper editing has been observed in several different cancers. Despite the significance of this form of regulation of RNA structure and function, there remain key gaps in our understanding of A to I RNA editing. In addition, given ADARs’ ability to change RNA sequence, there is growing interest in harnessing this property and directing it to correct disease-associated G-to-A mutations. Key questions in this field that will be addressed in this project are: 1) What are the structures of key protein-RNA complexes in editing pathways? Structures of full length human ADAR2 bound to different RNA substrates along with structures of ADAR1 bound to RNA are necessary for a full understanding of substrate recognition and selectivity in RNA editing. 2) Can we develop potent, selective and low MW ADAR inhibitors? Such inhibitors could serve as lead compounds in the development of ADAR1-targeted cancer therapies. 3) Can we develop new strategies to evolve mutant editing enzymes and novel substrate RNAs? The results of these efforts will inform the design of highly efficient and selective reagents for directed RNA editing applications. Our laboratory also has a long standing interest in the development of chemical modifications of RNA that can control the interaction with RNA-binding proteins. Much of our recent work in this area has focused on controlling the interaction of RNA with components of siRNA-triggered or miRNA- triggered gene silencing pathways. The use of the RNAi pathway to study gene function has become a powerful tool in molecular biology and has been exploited in the development of new therapeutics. However, specific issues exist that limit its application. These issues include off-target effects that arise from the ability of an siRNA guide strand to function as a miRNA. In addition, antisense oligonucleotides targeting miRNAs (anti-miRs) have significant therapeutic potential and require chemical modification for stability and efficacy. Up to this point, the development of new chemical modifications of therapeutic RNAs has been largely an ad hoc process. The key question addressed in this aspect of the proposed project is: Can we develop an effective systematic approach to new RNA modifications that modulate protein-RNA interactions in interference pathways? The immediate impact of these studies will be to provide new modifications to siRNAs and anti-miRs that improve potency and selectivity. However, our continued refinement of an approach that uses computational screening coupled with versatile RNA modification chemistry will be generally applicable other projects that involve chemically modified RNA for therapeutics.