Methods for Determination of 2′-O-Me in RNA

Abstract

Ribose methylation is one of the most abundant RNA modifications and is found in all kingdoms of life and all major classes of RNA (rRNA, tRNA, and mRNA). Ribose methylations are introduced by stand-alone enzymes or by generic enzymes guided to the target by small RNA guides. The most abundant mechanism of ribose methylation is found in rRNA of Archaea and Eukarya where a methyltransferase (fibrillarin) use sRNA (Archaea) or box C/D snoRNA (Eukarya) as guide RNAs to specify the site of modification. The general function of these modifications is to promote ribosome biogenesis, in particular folding of the ribosomal RNA. Furthermore, some modifications affect the fidelity of translation. The function of individual modifications has in many cases remained elusive, because genetic deletion of the modification has a weak phenotype or no phenotype at all. Another problem is that methods for mapping modifications and quantitating the fraction of RNA molecules modified in a population until recently remained poorly developed. Here, we review the methods that have been used to study 2′-O-Me in RNA starting with the original approach employing in vivo isotope labeling followed by paper chromatography. The next generation of methods typically addressed one nucleotide at a time and was mostly based on primer extension. Finally, more recent mass spectrometry and high-throughput sequencing methods hold promise to reveal a new biology of this widespread type of nucleotide modification.