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Biochemistry. 2013 Nov 12;52(45):7857-69. doi: 10.1021/bi4006539. Epub 2013 Oct 31.

RNA-Seq analysis identifies a novel set of editing substrates for human ADAR2 present in Saccharomyces cerevisiae.

Author information

1
Department of Chemistry, University of California , One Shields Avenue, Davis, California 95616, United States.

Abstract

ADAR2 is a member of a family of RNA editing enzymes found in metazoa that bind double helical RNAs and deaminate select adenosines. We find that when human ADAR2 is overexpressed in the budding yeast Saccharomyces cerevisiae it substantially reduces the rate of cell growth. This effect is dependent on the deaminase activity of the enzyme, suggesting yeast transcripts are edited by ADAR2. Characterization of this novel set of RNA substrates provided a unique opportunity to gain insight into ADAR2's site selectivity. We used RNA-Seq. to identify transcripts present in S. cerevisiae subject to ADAR2-catalyzed editing. From this analysis, we identified 17 adenosines present in yeast RNAs that satisfied our criteria for candidate editing sites. Substrates identified include both coding and noncoding RNAs. Subsequent Sanger sequencing of RT-PCR products from yeast total RNA confirmed efficient editing at a subset of the candidate sites including BDF2 mRNA, RL28 intron RNA, HAC1 3'UTR RNA, 25S rRNA, U1 snRNA, and U2 snRNA. Two adenosines within the U1 snRNA sequence not identified as substrates during the original RNA-Seq. screen were shown to be deaminated by ADAR2 during the follow-up analysis. In addition, examination of the RNA sequence surrounding each edited adenosine in this novel group of ADAR2 sites revealed a previously unrecognized sequence preference. Remarkably, rapid deamination at one of these sites (BDF2 mRNA) does not require ADAR2's dsRNA-binding domains (dsRBDs). Human glioma-associated oncogene 1 (GLI1) mRNA is a known ADAR2 substrate with similar flanking sequence and secondary structure to the yeast BDF2 site discovered here. As observed with the BDF2 site, rapid deamination at the GLI1 site does not require ADAR2's dsRBDs.

PMID:
24124932
PMCID:
PMC3856204
DOI:
10.1021/bi4006539
[Indexed for MEDLINE]
Free PMC Article

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