Method for Direct Mass-Spectrometry-Based Identification of Monomethylated RNA Nucleoside Positional Isomers and Its Application to the Analysis of Leishmania rRNA

RNA post-transcriptional modifications are common in
all kingdoms
of life and are predominantly affiliated with methylations at various
nucleobase positions. Methylations occur frequently at specific sites
on the RNA nucleobases and appear to regulate site-specific intermolecular/intramolecular
interactions. Herein, we present a method that utilizes liquid chromatography–mass
spectrometry (LC–MS) to identify positional monomethylated
RNA nucleoside isomers. The method produces profiles of in-source
fragmentation and subsequent tandem mass spectrometry (MS2) (pseudo-MS3) of RNase-digested fragments of an RNA and
distinguishes between positional methylated nucleobase isomers by
comparing their intranucleobase fragment ion profiles with signature
profiles derived from authentic isomers. For method validation, we
independently determined the positions of all known monomethylated
nucleoside isomers in the Escherichia coli 16S/23S rRNAs. As proof of concept, we further applied this technology
to fully characterize the base-modified nucleoside positional isomers,
in rRNAs derived from Leishmania donovani, a human blood parasite afflicting millions around the globe. The
method described herein will be highly beneficial for the delineation
of RNA modification profiles in various cellular RNAs, and as it only
requires a subpicomole amount of RNA, it could also be used for the
structure–function studies of RNA populations represented in
minute amounts in the cell.