Adenosine Deaminases that act on RNA (ADARs) are a family of RNA editing enzymes that convert adenosines to inosines in double-stranded RNA (dsRNA). ADARs are essential in mammals and required for the health of all animals. An important function of ADARs is to target codons to allow a single mRNA to give rise to multiple protein isoforms. Surprisingly, in all studied organisms, recoding editing sites are rare compared to the extensive, non-selective, editing that occurs in noncoding regions of mRNAs, such as introns and untranslated regions. The functions of non-selective editing are unknown, and the proposed research will capitalize on technological advances, such as RNAseq and CRISPR protocols, to fill this gap in knowledge. Studies will utilize the model organism C. elegans, since non-selective editing may be solely responsible for phenotypes of C. elegans lacking ADARs. Experiments will test a model whereby non-selective editing precludes Dicer processing of dsRNA, thus regulating gene silencing in a developmental and tissue-specific manner. Studies are facilitated by the genome-wide identification of Editing-Enriched Regions (EERs), which define the non-selectively edited dsRNAome. RNAseq analyses will define expression of EER-associated genes, and endogenous siRNAs (endo-siRNAs) matching these genes, to reveal genes that are good candidates for ADAR regulation of gene silencing. Deletion of the EER with CRISPR protocols will allow confirmation that ADAR regulation depends on the presence of the EER. Endo-siRNAs aligning to EERs have features of direct Dicer products, a species of small RNA not previously observed among C. elegans endo- siRNAs. Factors involved in the biogenesis of, and silencing by, this newly identified class of endo-siRNAs will be identified with a screen using a C. elegans strain expressing a GFP reporter that is silenced in an ADAR mutant strain. Proposed studies will yield strains that lack double-stranded EER(s) in specific genes, and ADAR mutant strains with additional deletions in factors that rescue ADAR-regulated silencing. These strains will be tested for their ability to rescue phenotypes of C. elegans lacking ADARs. While correlation of a phenotype to a specific gene will be a priority, a degron protocol will provide an alternate strategy. Studies are designed to provide long-awaited details about the functions of C. elegans ADARs, and pave the way for defining similarities and differences in roles of non-selective editing in other organisms.

Public Health Relevance

ADAR RNA editing enzymes are essential for human life, with established functions in 'editing' mRNA codons to allow multiple protein isoforms. However, recoding sites are rare, and there are far more editing sites in noncoding regions of mRNAs, but the functions of this editing are unknown. Experiments of this proposal are designed to reveal these functions, possibly revealing regulatory pathways important for innate immunity and gene silencing.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics B Study Section (MGB)
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Bender, Michael T
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University of Utah
Schools of Medicine
Salt Lake City
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Reich, Daniel P; Tyc, Katarzyna M; Bass, Brenda L (2018) C. elegans ADARs antagonize silencing of cellular dsRNAs by the antiviral RNAi pathway. Genes Dev 32:271-282
Blango, Matthew G; Bass, Brenda L (2016) Identification of the long, edited dsRNAome of LPS-stimulated immune cells. Genome Res 26:852-62
Whipple, Joseph M; Youssef, Osama A; Aruscavage, P Joseph et al. (2015) Genome-wide profiling of the C. elegans dsRNAome. RNA 21:786-800
Kuttan, Ashani; Bass, Brenda L (2012) Mechanistic insights into editing-site specificity of ADARs. Proc Natl Acad Sci U S A 109:E3295-304
Warf, M Bryan; Shepherd, Brent A; Johnson, W Evan et al. (2012) Effects of ADARs on small RNA processing pathways in C. elegans. Genome Res 22:1488-98
Bass, Brenda; Hundley, Heather; Li, Jin Billy et al. (2012) The difficult calls in RNA editing. Interviewed by H Craig Mak. Nat Biotechnol 30:1207-9
Eggington, Julie M; Greene, Tom; Bass, Brenda L (2011) Predicting sites of ADAR editing in double-stranded RNA. Nat Commun 2:319
Warf, M Bryan; Johnson, W Evan; Bass, Brenda L (2011) Improved annotation of C. elegans microRNAs by deep sequencing reveals structures associated with processing by Drosha and Dicer. RNA 17:563-77
Evan Johnson, W; Welker, Noah C; Bass, Brenda L (2011) Dynamic linear model for the identification of miRNAs in next-generation sequencing data. Biometrics 67:1206-14
Hundley, Heather A; Bass, Brenda L (2010) ADAR editing in double-stranded UTRs and other noncoding RNA sequences. Trends Biochem Sci 35:377-83

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