Data-Independent Acquisition for the Detection of Mononucleoside RNA Modifications by Mass Spectrometry

Abstract

RNA is dynamically modified in cells by a plethora of chemical moieties to modulate molecular functions and processes. Over 140 modifications have been identified across species and RNA types, with the highest density and diversity of modifications found in tRNA (tRNA). The methods used to identify and quantify these modifications have developed over recent years and continue to advance, primarily in the fields of next-generation sequencing (NGS) and mass spectrometry (MS). Most current NGS methods are limited to antibody-recognized or chemically derivatized modifications and have limitations in identifying multiple modifications simultaneously. Mass spectrometry can overcome both of these issues, accurately identifying a large number of modifications in a single run. Here, we present advances in MS data acquisition for the purpose of RNA modification identification and quantitation. Using this approach, we identified multiple tRNA wobble position modifications in Arabidopsis thaliana that are upregulated in salt-stressed growth conditions and may stabilize translation of salt stress induced proteins. This work presents improvements in methods for studying RNA modifications and introduces a possible regulatory role of wobble position modifications in A. thaliana translation.

Identification of a novel 5-aminomethyl-2-thiouridine methyltransferase in tRNA modification

“…Interestingly, the presence of mnm-modified uridines has been reported in tRNAs of certain Gram-positive bacteria and plants ( 36–41 ). Modern liquid chromatography–mass spectrometry (LC–MS)-based analyses of cellular tRNAs have confirmed that the modifications are distributed in Gram-positive bacteria like Bacillus subtilis ( 42 , 43 ) and Staphylococcus aureus ( 44 ) and plant chloroplasts such as Arabidopsis thaliana ( 45 ) and Oryza sativa ( 46 ). These studies suggest that an MnmC-like activity must be present in those organisms.…”

SWAMNA: a comprehensive platform for analysis of nucleic acid modifications

“…13 Previously, we found that RNA can be efficiently digested using the "one-pot" reaction by mixing the nuclease P1 (NP1), phosphodiesterase-I (PDE-I), phosphodiesterase II (PDE-II), and alkaline phosphatase (AP). 11 At the same time, Wang and co-workers demonstrated the workflow for DNA digestion. 14 Therefore, we incorporated the https://doi.org/10.26434/chemrxiv-2023-kdb28 ORCID: https://orcid.org/0000-0002-8512-6053 Content not peer-reviewed by ChemRxiv.…”

“…Recently, our lab and the Sabido ´group applied data-independent acquisition (DIA) to enable the potential detection of unanticipated molecular species. 14,15 These advances captured much attention in using LC-MS/MS to analyze nucleic acid modifications; however, the absence of an comprehensive platform for the characterization of DNA and RNA modifications hampers its potential for broader applications.…”

SWAMNA: a comprehensive platform for analysis of nucleic acid modifications

“…Recently, our lab and the Sabidó group applied dataindependent acquisition (DIA) to enable the potential detection of unanticipated molecular species. 11,12 These advances captured much attention in using LC-MS/MS to analyze nucleic acid modifications; however, the absence of an comprehensive platform for the characterization of DNA and RNA modifications hampers its potential for broader applications.…”