A mass spectrometry-based method for comprehensive quantitative determination of post-transcriptional RNA modifications: the complete chemical structure ofSchizosaccharomyces pomberibosomal RNAs

Abstract

We present a liquid chromatography–mass spectrometry (LC-MS)-based method for comprehensive quantitative identification of post-transcriptional modifications (PTMs) of RNA. We incorporated an in vitro-transcribed, heavy isotope-labeled reference RNA into a sample RNA solution, digested the mixture with a number of RNases and detected the post-transcriptionally modified oligonucleotides quantitatively based on shifts in retention time and the MS signal in subsequent LC-MS. This allowed the determination and quantitation of all PTMs in Schizosaccharomyces pombe ribosomal (r)RNAs and generated the first complete PTM maps of eukaryotic rRNAs at single-nucleotide resolution. There were 122 modified sites, most of which appear to locate at the interface of ribosomal subunits where translation takes place. We also identified PTMs at specific locations in rRNAs that were altered in response to growth conditions of yeast cells, suggesting that the cells coordinately regulate the modification levels of RNA.

Mapping of Complete Set of Ribose and Base Modifications of Yeast rRNA by RP-HPLC and Mung Bean Nuclease Assay

“…Although these techniques have been successful in providing the preliminary information, they still lack the sensitivity and quantitative information as compared with the state of art technologies like RP-HPLC and mass spectrometry [18–20]. …”

Mapping Post‐Transcriptional Modifications onto Transfer Ribonucleic Acid Sequences by Liquid Chromatography Tandem Mass Spectrometry

“…By differentially labeling the RNase digestion products from the two samples using various heavy isotope strategies [62,63,64,65], digestion products that are equivalent between the two samples will appear as doublets in the LC-MS/MS data. Missing doublets (i.e., singlets) occur when the underlying tRNA sequences differ between the two samples or when the two samples are modified differently (Figure 6).…”

Mass spectrometry of modified RNAs: recent developments

“…48, 49 Figure 3 illustrates the former, wherein the base composition of the RNase digestion product can be determined based on the m/z measurement and then constrained by the number of carbons or nitrogens present, which is determined by the mass shift induced by the metabolic label. 47 Alternatively, metabolic labeling to deplete heavy isotopes simplifies detection and identification of larger molecular weight RNase digestion products, and has been used to improve the CARD approach for identification of modified RNAs (Figure 4).…”