The RNA editing ADAR enzymes convert adenosines to inosines in RNA. This phenomenon is widespread with > 15,000 A to I sites known in human transcripts. Since inosine is decoded as guanosine during translation, this modification can lead to changes in the meaning of codons (recoding). There are at least 50 different A to I sites in human mRNAs that cause recoding and recoding is common in the nervous system. Indeed, ADARs are necessary for a properly functioning nervous system and are known to regulate behavior in metazoans. Mutations in the human ADAR1 gene cause the skin disorder Dyschromatosis Symmetrica Hereditaria (DSH) and the autoimmune disease Aicardi-Goutieres Syndrome (AGS). Also, hyper editing has been observed at certain sites in cancer cells, such as in the mRNA for AZIN1 (antizyme inhibitor 1). In contrast however, hypo editing is observed in the glioma- associated ongene 1 (Gli1), a transcription factor important in the Hedgehog signaling pathway considered a therapeutic target in several types of cancer. Despite the significance of this form of regulation of RNA structure and function, our understanding of the mechanism of A to I editing is deficient. For instance, the selectivity for specific adenosines within ADAR substrates remains difficult to fully explain. Furthermore, pharmacological methods for controlling RNA editing at specific sites do not currently exist. In this competitive renewal o an R01 project, we will address these knowledge gaps through the application of nucleic acid chemistry coupled with techniques from structural biology and biochemistry. The results of these studies will extend our basic understanding of the process of RNA editing as well as lead to new methods for its control. Given our laboratory's recent discovery of small RNAs that react rapidly with isolated ADAR deaminase domains, we are in a position like never before to provide important information about the ADAR-RNA interaction. Thus, we will pursue X-ray diffraction quality crystals of ADAR deaminase domains in complex with RNAs derived from sequences shown by us to react rapidly and solve the structures in collaboration with Professor Andrew Fisher. We expect the resulting structures to reveal details of the ADAR-RNA interaction that will shed light on the observed editing site selectivity. In addition, we will directly test two competig hypotheses for the basis of editing site selectivity. In addition, to advance our goal of developin methods to control editing pharmacologically, we will generate new antisense oligonucleotides capable of regulating RNA editing in a site-specific manner (editing inhibitors and editing enhancers). The resulting compounds will be useful tools to probe the mechanism of the editing reaction, the biological function of specific editing events and could have long- term therapeutic potential. Finally, we will provide a deeper understanding of the ADAR reaction by identifying sites in the catalytic domain important for catalysis not previously appreciated by carrying out directed evolution to convert the hADAR3 catalytic domain into a functional RNA editing enzyme.

Public Health Relevance

RNA editing catalyzed by the ADAR enzymes is a form of control of gene expression that is perturbed in a variety of human diseases including Dyschromatosis Symmetrica Hereditaria (DSH), Aicardi-Goutierres Syndrome (AGS), epilepsy, depression and cancer. The proposed studies will extend our basic understanding of the process of RNA editing as well as lead to new methods for its control.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM061115-14
Application #
8964627
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Barski, Oleg
Project Start
2001-03-01
Project End
2019-07-31
Budget Start
2015-08-01
Budget End
2016-07-31
Support Year
14
Fiscal Year
2015
Total Cost
$305,154
Indirect Cost
$89,616
Name
University of California Davis
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Zheng, Yuxuan; Lorenzo, Claire; Beal, Peter A (2017) DNA editing in DNA/RNA hybrids by adenosine deaminases that act on RNA. Nucleic Acids Res 45:3369-3377
Thomas, Justin M; Beal, Peter A (2017) How do ADARs bind RNA? New protein-RNA structures illuminate substrate recognition by the RNA editing ADARs. Bioessays 39:
Fisher, Andrew J; Beal, Peter A (2017) Effects of Aicardi-Goutières syndrome mutations predicted from ADAR-RNA structures. RNA Biol 14:164-170
Wang, Yuru; Beal, Peter A (2016) Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method. Nucleic Acids Res 44:9872-9880
Matthews, Melissa M; Thomas, Justin M; Zheng, Yuxuan et al. (2016) Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity. Nat Struct Mol Biol 23:426-33
Kuhn, Claus-D; Wilusz, Jeremy E; Zheng, Yuxuan et al. (2015) On-enzyme refolding permits small RNA and tRNA surveillance by the CCA-adding enzyme. Cell 160:644-58
Wang, Yuru; Havel, Jocelyn; Beal, Peter A (2015) A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1. ACS Chem Biol 10:2512-9
Mizrahi, Rena A; Shin, Dongwon; Sinkeldam, Renatus W et al. (2015) A Fluorescent Adenosine Analogue as a Substrate for an A-to-I RNA Editing Enzyme. Angew Chem Int Ed Engl 54:8713-6
Phelps, Kelly J; Tran, Kiet; Eifler, Tristan et al. (2015) Recognition of duplex RNA by the deaminase domain of the RNA editing enzyme ADAR2. Nucleic Acids Res 43:1123-32
Phelps, Kelly J; Ibarra-Soza, José M; Tran, Kiet et al. (2014) Click modification of RNA at adenosine: structure and reactivity of 7-ethynyl- and 7-triazolyl-8-aza-7-deazaadenosine in RNA. ACS Chem Biol 9:1780-7

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