A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA

Abstract

Here we describe an analytical platform for systems-level quantitative analysis of modified ribonucleosides in any RNA species, with a focus on stress-induced reprogramming of tRNA as part of a system of translational control of cell stress response. The chapter emphasizes strategies and caveats for each of the seven steps of the platform workflow: 1) RNA isolation, 2) RNA purification, 3) RNA hydrolysis to individual ribonucleosides, 4) chromatographic resolution of ribonucleosides, 5) identification of the full set of modified ribonucleosides, 6) mass spectrometric quantification of ribonucleosides, 6) interrogation of ribonucleoside datasets, and 7) mapping the location of stress-sensitive modifications in individual tRNA molecules. We have focused on the critical determinants of analytical sensitivity, specificity, precision and accuracy in an effort to ensure the most biologically meaningful data on mechanisms of translational control of cell stress response. The methods described here should find wide use in virtually any analysis involving RNA modifications.

tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence

“…Using chromatography-coupled mass spectrometry (LC-MS)212223, we identified 40 distinct ribonucleoside modifications in purified tRNA (Fig. 1a, parameters in Supplementary Data 1).…”

“…All mass spectrometers were operated in positive ion mode. Relative quantification of tRNA modifications was performed as previously described2223.…”

Small RNA Modifications: Integral to Function and Disease

“…While LC-MS/MS is a powerful approach for quantifying the compositional changes of RNA modifications, this method requires digestion of RNAs into short segments or single nucleosides before detection, therefore making it difficult to unambiguously assign modifications to specific transcripts (Figure 4A) [51,66,67]. Thus, complementary approaches to map RNA modifications to specific RNAs are required to gain a more comprehensive view of modified small RNAs.…”

“…For example, m 1 A antibodies can detect tRNA conformational changes and circulating tsRNAs that arise from acute cellular stress, potentially providing an earlier indicator of tissue injury than other markers of apoptosis and DNA damage (Figure 3A) [65]. High-throughput liquid chromatography–tandem mass spectrometry (LC-MS/MS) provides a complementary approach that can be used for quantification of multiple RNA modifications within a single RNA sample [40,51,66–68]. LC-MS/MS has uncovered a diversity of previously overlooked RNA modifications among small RNAs (10–30 nt, 30–60 nt) in mouse liver [51], revealing dynamic changes for multiple RNA modifications in mouse models of diabetes (Gm, m 5 Cm, Cm, Am, Um) [51] (Figure 1B).…”

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

“…Thus, although the RNA isolation process can be flexible, caution is advised to ensure efficient isolation of pure, high quality tRNA samples. This can be evaluated using UV absorbance, denaturing gel electrophoresis, or fluorescent dye-based electrophoresis [116]. …”