RNA oxidation mm-miR and sc-mRNA

RNA oxidation is an important, though little understood process, largely overshadowed by research on DNA. A clear shortcoming is the lack of methods precisely locating oxidized residues in RNA sequences. Development of corresponding methods is hampered by the diversity of occurring oxidation products as well as by their diffuse distribution and low stoichiometry. Here we develop a novel deep sequencing method termed OAbSeq that maps oxidized sites with high sensitivity by exploiting aniline-induced strand scission at noncanonical nucleoside structures. This approach generates ligation-competent fragments that serve as highly specific molecular entry points for library preparation, yielding a high signal-to-noise ratio. Application to yeast RNA, resulted in the detection of widespread but low amplitude signals predominantly at purine sites, especially at guanosines. To elucidate the molecular genesis of these signals, we treated yeast cells with hypochlorous acid, a cell-penetrating exogenously added oxidant, which increased signal intensity while maintaining the general pattern at guanosine residues. Side-by-side quantification of 8-oxoguanosine (oxo8G) and abasic sites revealed that abasic sites were more abundant than oxo8G following oxidative treatment in vitro and under physiological conditions. Our data indicate that RNA oxidation at guanosines proceeds via transient oxo8G and results primarily in abasic sites, which constitute the predominant molecular oxidation product that can be mapped with nucleotide precision by the new OAbSeq. We posit that abasic sites can serve as a superior marker for oxidative damage of RNA than oxo8G, opening new avenues for understanding RNA oxidation dynamics in cells