RNA marker modifications reveal the necessity for rigorous preparation protocols to avoid artifacts in epitranscriptomic analysis

Abstract

The accurate definition of an epitranscriptome is endangered by artefacts resulting from RNA degradation after cell death, a ubiquitous yet little investigated process. By tracing RNA marker modifications through tissue preparation protocols, we identified a major blind spot from daily lab routine, that has massive impact on modification analysis in small RNAs. In particular, m6,6A and Am as co-varying rRNA marker modifications, appeared in small RNA fractions following rRNA degradation in vitro and in cellulo. Analysing mouse tissue at different time points post mortem, we tracked the progress of intracellular RNA degradation after cell death, and found it reflected in RNA modification patterns. Differences were dramatic between liver, where RNA degradation commenced immediately after death, and brain, yielding essentially undamaged RNA. RNA integrity correlated with low amounts of co-varying rRNA markers. Thus validated RNA preparations featured differentially modified tRNA populations whose information content allowed a distinction even among the related brain tissues cortex, cerebellum and hippocampus. Inversely, advanced cell death correlated with high rRNA marker content, and correspondingly little with the naïve state of living tissue. Therefore, unless RNA and tissue preparations are executed with utmost care, interpretation of modification patterns in tRNA and small RNA are prone to artefacts.

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

“…To address this question, we isolated total RNA along the growth of E. coli, used two mRNA enrichment methods, electrophoretic separation by size and rRNA depletion, followed by stringent validation of RNA identity and analyzed the individual fractions by isotope dilution nucleoside mass spectrometry (LC-MS/MS). Similar to a recent study in mice, [19] our systematic study reveals the challenges connected to a size-dependent purification of mRNA fractions but also highlights the benefits of an unbiased assessment of the chemical diversity of bacterial RNAs. We could identify RNA modifications which appear to be involved in bacterial stress response.…”

“…The observation of m 6,6 A and m 5 C is in agreement with a recent report from the Helm lab, where small RNA fractions gained through sizeselection are contaminated with rRNA fragments and the latter has been identified as the source of rRNA specific modifications in mouse tRNA. [19] As E. coli has no m 5 C reported in its tRNA, but in both rRNAs, we suggest that the m 5 C signal might be also rRNA fragment derived. The m 1 A signal might be related to a direct methylation of adenosine in tRNA upon exposure to natural methylating agents.…”

“…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.…”

Experimental and computational workflow for the analysis of tRNA pools from eukaryotic cells by mim-tRNAseq

“… Timing: days (depending on the organism, developmental stage, and cell or tissue type) Culture cells in an appropriate medium or collect tissue samples according to the purpose of the experiment. CRITICAL: Extra care should be taken when working with animals, as post-mortem RNA degradation can occur rapidly in some tissue types ( Richter et al., 2022 ). Dissection should be performed as quickly as possible, and tissue samples should be snap-frozen in liquid nitrogen and stored at −80°C.…”

“… CRITICAL: Extra care should be taken when working with animals, as post-mortem RNA degradation can occur rapidly in some tissue types ( Richter et al., 2022 ). Dissection should be performed as quickly as possible, and tissue samples should be snap-frozen in liquid nitrogen and stored at −80°C.…”