The RNA editing ADAR enzymes convert adenosines to inosines in RNA. 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. Recoding is common in the nervous system with targets including ligand-gated ion channels, voltage-gated ion channels and G-protein coupled receptors. Indeed, ADARs are necessary for a properly functioning nervous system and are known to regulate behavior in metazoans. However, little is known about the effect of recoding for targets with roles outside the nervous system. Perturbations in A to I editing have been observed in several human diseases including amyotrophic lateral sclerosis (ALS), dyschromatosis symmetrica hereditaria (DSH), Prader-Willi syndrome (PWS), epilepsy, depression and cancer. A recent study also implicates ADARs in the control of aging. Despite the significance of this form of epigenetics, our understanding of the mechanism and regulation of A to I editing is deficient. For instance, the selectivity for specific adenosines within ADAR substrates remains difficult to fully explain due to a lack of detailed characterization for ADAR-RNA complexes. Furthermore, pharmacological methods for controlling RNA editing do not currently exist limiting the types of studies possible to probe its biological function. In this competitive renewal of an R01 project, these knowledge gaps will be addressed through the application of synthetic chemistry coupled with techniques from molecular biology and biochemistry. The results of these studies will extend our basic understanding of the process of RNA editing and its effects on protein function as well as lead to new methods for its control. Methods for site-selective inhibition of RNA editing will be developed. Backbone modified antisense oligonucleotides and helix- threading peptoids that target RNA editing substrates will be investigated for this purpose. In addition, we will define factors controlling editing selectivity and mechanisms of inhibition for the ADAR2 reaction. A functional screen will be used to define the importance in controlling editing site selectivity of the length and sequence in a linker structure between two ADAR2-RNA interaction domains. In addition, ADAR2 mutants containing unnatural amino acids and RNA containing nucleoside analogs will be used to map ADAR2-RNA interactions. A novel genetic selection will identify cyclic peptide inhibitors of ADAR2 and follow-up studies with these inhibitors will identify points in the ADAR2 reaction susceptible to small molecule control. In addition, we will define the basis for ADAR1 editing of the mRNA for the DNA repair enzyme NEIL1. These efforts will define structure/activity relationships for the ADAR1 reaction and extend our understanding of the recoding of targets with functions outside the nervous system. Finally, this project will produce new molecules for crystallization trials of ADAR-RNA complexes.

Public Health Relevance

RNA editing catalyzed by the ADAR enzymes is a form of epigenetic control of gene expression that is perturbed in a variety of human diseases including amyotrophic lateral sclerosis (ALS), dyschromatosis symmetrica hereditaria (DSH), Prader-Willi syndrome (PWS), 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)
Type
Research Project (R01)
Project #
5R01GM061115-13
Application #
8635357
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Barski, Oleg
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California Davis
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Davis
State
CA
Country
United States
Zip Code
95618
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Mizrahi, Rena A; Schirle, Nicole T; Beal, Peter A (2013) Potent and selective inhibition of A-to-I RNA editing with 2'-O-methyl/locked nucleic acid-containing antisense oligoribonucleotides. ACS Chem Biol 8:832-9
Eifler, Tristan; Pokharel, Subhash; Beal, Peter A (2013) RNA-Seq analysis identifies a novel set of editing substrates for human ADAR2 present in Saccharomyces cerevisiae. Biochemistry 52:7857-69
Phelps, Kelly; Morris, Alexi; Beal, Peter A (2012) Novel modifications in RNA. ACS Chem Biol 7:100-9
Goodman, Rena A; Macbeth, Mark R; Beal, Peter A (2012) ADAR proteins: structure and catalytic mechanism. Curr Top Microbiol Immunol 353:1-33
Schirle, Nicole T; Goodman, Rena A; Krishnamurthy, Malathy et al. (2010) Selective inhibition of ADAR2-catalyzed editing of the serotonin 2c receptor pre-mRNA by a helix-threading peptide. Org Biomol Chem 8:4898-904
Yeo, Jongchan; Goodman, Rena A; Schirle, Nicole T et al. (2010) RNA editing changes the lesion specificity for the DNA repair enzyme NEIL1. Proc Natl Acad Sci U S A 107:20715-9
Wang, Selina C; Beal, Peter A; Tantillo, Dean J (2010) Covalent hydration energies for purine analogs by quantum chemical methods. J Comput Chem 31:721-5
Jayalath, Prasanna; Pokharel, Subhash; Veliz, Eduardo et al. (2009) Synthesis and evaluation of an RNA editing substrate bearing 2'-deoxy-2'-mercaptoadenosine. Nucleosides Nucleotides Nucleic Acids 28:78-88
Pokharel, Subhash; Jayalath, Prasanna; Maydanovych, Olena et al. (2009) Matching active site and substrate structures for an RNA editing reaction. J Am Chem Soc 131:11882-91

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