GSE109925
Conserved Methyltransferase Spb1 Targets mRNAs for Regulated Modification with 2'-O-Methyl Ribose [meTH-Seq]
Non-coding RNAs contain dozens of chemically distinct modifications, of which only a few have been identified in mRNAs. The recent discovery that certain tRNA modifying enzymes also target mRNAs suggests the potential for many additional mRNA modifications. Here, we show that conserved tRNA 2'-O-methyltransferases Trm3, 7,13 and 44, and rRNA 2'-O-methyltransferase Spb1, interact with specific mRNA sites in yeast by crosslinking immunoprecipitation and sequencing (CLIP-seq). We developed sequencing of methylation at two prime hydroxyls (MeTH-seq) for transcriptome-wide mapping of 2'-O-methyl ribose (Nm) with single-nucleotide resolution, and discover thousands of potential Nm sites in mRNAs. Genetic analysis identified hundreds of mRNA targets for the Spb1 methyltransferase, which can target both mRNA and non-coding RNA for environmentally regulated modification. Our work identifies Nm as a prevalent mRNA modification that is likely to be conserved and provides methods to investigate its distribution and regulation. Overall design: Each experiment consists of two libraries, one reverse transcribed in low (restrictive) Mg2+ and one at permissive concentrations. Nm sites are identified through peaks, ie. positions where there are more reverse transcription stops in the restrictive than in the permissive condition. We include data for 41 such experiments across two growth conditions, and in various knockout backgrounds.