Functional Characterization of 2-O-Methylation and Pseudouridylation Guide RNAs

Pseudouridines and 2′-O -methylated nucleotides are ubiquitous constituents of stable cellular RNAs. In eukaryotes, posttranscriptional synthesis of most pseudouridines and 2′-O -methylated nucleotides is directed by sequence-specific guide RNAs (gRNAs). In recent years, an enormous number of novel putative modification gRNAs have been identified in a broad variety of organisms by using bioinformatics and large-scale cDNA-sequencing approaches. Understanding of the function of the novel modification gRNAs as well as the functional importance of modified nucleotides requires techniques that support the site-specific detection of 2′-O -methylated nucleotides and pseudouridines. Here, we describe rapid, reverse transcription-based methods to map 2′-O -methylated nucleotides and pseudouridines in any cellular RNA.