RT Journal Article
SR Electronic
T1 Dissecting the basis for differential substrate specificity of ADAR1 and ADAR2
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2023.01.16.524339
DO 10.1101/2023.01.16.524339
A1 Marlon S. Zambrano-Mila
A1 Monika Witzenberger
A1 Anna Uzonyi
A1 Ronit Nir
A1 Shay Ben-Aroya
A1 Erez Y. Levanon
A1 Schraga Schwartz
YR 2023
UL http://biorxiv.org/content/early/2023/01/17/2023.01.16.524339.abstract
AB Millions of adenosines are deaminated throughout the transcriptome by ADAR1 and ADAR2, modulating double-stranded RNA (dsRNA) immunogenicity and recoding mRNA. The high variability in the susceptibility of different adenosines to editing begs the question of what are the determinants of substrate specificity. Here, we systematically monitor how secondary structure modulates ADAR2 vs ADAR1 substrate selectivity, on the basis of systematic probing of thousands of synthetic sequences transfected into ADAR1-deleted cell lines exogenously expressing either ADAR2 or ADAR1. In both cases, structural disruptions gave rise to symmetric, strand-specific induced editing at a fixed offset, but of varying length: -26 nt for ADAR2, and -35 nt for ADAR1. We dissect the basis for the differences in offset between ADAR1 and ADAR2 via diverse mutants, domain-swaps, and ADAR evolutionary homologs, and reveal that it is encoded by the differential RNA binding domain architecture. We demonstrate that this offset-enhanced editing can allow an improved design of ADAR2-recruiting therapeutics, with proof-of-concept experiments suggestive of increased on-target and potentially decreased off-target editing. Our findings provide novel insight into the determinants guiding ADAR2 substrate selectivity and into the roles of the RNA binding domains of ADAR1 and ADAR2 in mediating differential targeting, and should facilitate the design of improved ADAR-recruiting therapeutics.Competing Interest StatementThe authors have declared no competing interest.