Transcriptome-wide Profiling of Multiple RNA Modifications Simultaneously at Single-base Resolution

We report RBS-Seq, a new RNA bisulfite sequencing method enabling the sensitive and simultaneous detection of m5C, Y, and m1A at single-base resolution transcriptome-wide. For each, we detect ‘signature’ base mismatches (for m5C and m1A), or 1-2 base deletions (for Y) structurally explained by ribose ring-opened Y-mono-bisulfite adducts. Our profiles for Y reveal clear signatures at known sites in tRNAs and rRNAs, and provide hundreds of new targets in non-coding RNAs and mRNAs. However, our results diverge greatly from prior work, suggesting ~10-fold fewer m5C sites, and the absence of substantial m1A in mRNAs.