GSE109926
Conserved Methyltransferase Spb1 Targets mRNAs for Regulated Modification with 2'-O-Methyl Ribose [eCLIP]
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: For each protein of interest, we tag it so that it may be pulled down with an antibody, and prepare libraries from the associated RNA. We also prepare a no-pulldown size-matched input control for each protein.