A previously uncharacterized double-stranded RNA (dsRNA) unwinding activity has been reported to exist in Xenopus laevis, Caenorhabditis elegans and a number of mammalian tissue culture cells. The activity exhibits an unusual mechanism of unwinding: during the reaction adenosine residues are covalently modified to inosine residues. The covalent modification results in the substitution of a stable AU base-pair by the considerably less stable IU base-pair; thus the dsRNA substrates are permanently unwound. The long term goal of the proposed research is to determine the biological function of the unwinding/modifying activity. Although the role of the modification may be to unwind the dsRNA, the discovery that the activity covalently modifies its RNA substrate raises the possibility that the primary function of the activity is to modify rather than unwind. In regards to the long term goal, studies will focus on determining the natural biological substrate(s) of the unwinding/modifying activity. Given the ubiquity of double-stranded regions (intramolecular as well as intermolecular) within cellular RNAs, many potential substrates can be imagined. Experiments that will narrow the range of possibilities will be performed. The substrate specificity of the unwinding/modifying activity will be defined in more detail, utilizing previously developed in vitro assays. In addition, cellular RNAs will be analyzed to determine if a particular subset of RNAs is more likely to contain the biological substrate(s). Sensitive HPLC methods, as well as anti-inosine antibody, will be used to assay various populations of RNAs for the presence of inosine and, using an antibody to the unwinding/modifying activity, the ability of specific RNAs to interact with the activity will be tested. At later stages of the proposed research, microinjection techniques will be utilized to alter the expression of the unwinding/modifying activity in Xenopus oocytes and early embryos, and to monitor phenotypic changes, in particular changes in cellular RNAs, that may occur during altered expression. The proposal includes protocols for purifying the unwinding/modifying activity, raising antibodies to the protein and cloning the cDNA.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Biology Study Section (MBY)
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University of Utah
Schools of Medicine
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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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