Strategies to Avoid Artifacts in Mass Spectrometry‐Based Epitranscriptome Analyses

Abstract

In this report, we perform structure validation of recently reported RNA phosphorothioate (PT) modifications, a new set of epitranscriptome marks found in bacteria and eukaryotes including humans. By comparing synthetic PT‐containing diribonucleotides with native species in RNA hydrolysates by high‐resolution mass spectrometry (MS), metabolic stable isotope labeling, and PT‐specific iodine‐desulfurization, we disprove the existence of PTs in RNA from E. coli, S. cerevisiae, human cell lines, and mouse brain. Furthermore, we discuss how an MS artifact led to the initial misidentification of 2′‐O‐methylated diribonucleotides as RNA phosphorothioates. To aid structure validation of new nucleic acid modifications, we present a detailed guideline for MS analysis of RNA hydrolysates, emphasizing how the chosen RNA hydrolysis protocol can be a decisive factor in discovering and quantifying RNA modifications in biological samples.

Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

“…Further, our data suggest that ALKBH1 can install f 5 C on polyadenylated RNA. Notably, while bulk poly(A)RNA fractions can be contaminated with modified nucleosides from more abundant RNA species 35 , given the low levels of f 5 C in total RNA and the purity of our poly(A) fraction (Supplementary Fig. 19 ), we believe that the observed modification levels are unlikely to have originated from non-poly(A)RNAs.…”

Challenges with Simulating Modified RNA: Insights into Role and Reciprocity of Experimental and Computational Approaches

“…Through -omics methodology, MS can provide these data via high throughput technologies for larger RNAs as well as heterogeneous biological samples (cell lysates, etc.) [ 112 , 113 , 114 ]. Additionally, advances have been made in native MS to provide tertiary contact information as well as the stability of certain folded RNAs [ 115 , 116 , 117 , 118 ].…”

Opportunities and Challenges to Profile mRNA Modifications in Escherichia coli**

“…Systematic and careful studies have also revealed the potential pitfalls and provided guidelines for avoiding them. [34,19,35,36] For prokaryotes, the number of studies is currently small, but given the potential of the epitranscriptome for bacterial fitness, stress adaptation and thus human health in the context of bacterial infection, more studies will follow. Therefore, we set out to systematically test different methods for mRNA preparation using the model bacterium E. coli MG1655.…”

“…Recent advances in the analysis of RNA modifications by sequencing and mass spectrometry have led to an ever‐rising number of studies focusing on the molecular mechanism of mRNA modifications in eukaryotes. Systematic and careful studies have also revealed the potential pitfalls and provided guidelines for avoiding them [34,19,35,36] . For prokaryotes, the number of studies is currently small, but given the potential of the epitranscriptome for bacterial fitness, stress adaptation and thus human health in the context of bacterial infection, more studies will follow.…”