The Profile and Dynamics of RNA Modifications in Animals

Abstract

More than a hundred distinct modified nucleosides have been identified in RNA, but little is known about their distribution across different organisms, their dynamic nature and their response to cellular and environmental stress. Mass‐spectrometry‐based methods have been at the forefront of identifying and quantifying modified nucleosides. However, they often require synthetic reference standards, which do not exist in the case of many modified nucleosides, and this therefore impedes their analysis. Here we use a metabolic labelling approach to achieve rapid generation of bio‐isotopologues of the complete Caenorhabditis elegans transcriptome and its modifications and use them as reference standards to characterise the RNA modification profile in this multicellular organism through an untargeted liquid‐chromatography tandem high‐resolution mass spectrometry (LC‐HRMS) approach. We furthermore show that several of these RNA modifications have a dynamic response to environmental stress and that, in particular, changes in the tRNA wobble base modification 5‐methoxycarbonylmethyl‐2‐thiouridine (mcm5s2U) lead to codon‐biased gene‐expression changes in starved animals.

Determination of the presence of 5-methylcytosine in Paramecium tetraurelia

“…Positive controls together with Dev1 genomic DNA samples were sent to Storm Therapeutics Limited where they were enzymatically digested to individual nucleosides according to a previously optimised protocol[ 33 ]. The samples were analysed in both full-scan- and PRM-mode in an Orbitrap QExactive-HF High-Resolution Mass Spectrometer (Thermo Fisher, Waltham, Massachusetts, USA).…”

Modeling Meets Metabolomics—The WormJam Consensus Model as Basis for Metabolic Studies in the Model Organism Caenorhabditis elegans

“…These nucleotides are found in RNA and represent degradation products if found in free form. These modified bases have been measured by LC-MS from RNA samples in C. elegans (van Delft et al, 2017 ). These modifications play important roles in fine tuning RNA function, e.g., tRNA contains several modified nucleotides.…”

Function by Structure: Spotlights on Xist Long Non-coding RNA

“…The combinatorial complexity of amino acid chains is matched by the number of possible modifications of the nucleotides, which have been described as existing in at least 100 post-transcriptionally modified states (van Delft et al, 2017 ). The post-transcriptional modifications can potentially be reversed during the life-span of the RNA, giving the primary sequence a dynamic nature.…”

“…Recently, the N6 methylation of adenosine (m6A) was widely characterized in the transcriptome, and its functional role emerged in many cellular contexts (Dominissini et al, 2012 ; Meyer et al, 2012 ). m6A has been shown to be associated with mRNA stability; it also dictates lncRNA-protein accessibility and specificity (Dominissini et al, 2012 ; Ke et al, 2017 ; van Delft et al, 2017 ). Xist RNA was shown to be methylated in a handful of well-defined positions (Dominissini et al, 2012 ; Patil et al, 2016 ).…”