Identification of rare pseudouridylated microRNA by comprehensive smallRNA bisulfite sequencing of mouse and human tissues

Pseudouridine (Ψ) is an important post-transcriptional modification of many noncoding RNAs that is under-characterized in microRNA (miRNA) due to historical limitations in pseudouridine mapping methods. Ψ modification stabilizes RNA duplex structures and could therefore play an important role in miRNA target binding and repression. To investigate the extent to which mammalian miRNAs are modified with Ψ, we profiled the modification landscape of short (<30 nt) RNA in human cells and mouse tissues using bisulfite sequencing. Our approach was powered to detect small RNA pseudouridylation based on robust detection of known Ψ positions in tRNA fragments (tRFs), some of which show tissue-specific patterns of modification. In contrast with tRFs, we find that miRNA pseudouridylation is exceedingly rare, with a single modified miRNA (miR-3068-5p) identified in mouse tissues. Pseudouridylated miR-3068-5p diSerentially repressed predicted miRNA targets with less stable miRNA:mRNA pairing modes. This study fills a long-standing gap in transcriptome-wide Ψ profiling and reveals a new potential function for Ψ as a modulator of activity of small regulatory RNAs.