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.
RNA modifications are a well-recognized way of gene expression regulation at the post-transcriptional level. Despite the importance of this level of regulation, current knowledge on modulation of tRNA modification status in response to stress conditions is far from being complete. While it is widely accepted that tRNA modifications are rather dynamic, such variations are mostly assessed in terms of total tRNA, with only a few instances where changes could be traced to single isoacceptor species. Using Escherichia coli as a model system, we explored stress-induced modulation of 2′-O-methylations in tRNAs by RiboMethSeq. This analysis and orthogonal analytical measurements by LC-MS show substantial, but not uniform, increase of the Gm18 level in selected tRNAs under mild bacteriostatic antibiotic stress, while other Nm modifications remain relatively constant. The absence of Gm18 modification in tRNAs leads to moderate alterations in E. coli mRNA transcriptome, but does not affect polysomal association of mRNAs. Interestingly, the subset of motility/chemiotaxis genes is significantly overexpressed in ΔTrmH mutant, this corroborates with increased swarming motility of the mutant strain. The stress-induced increase of tRNA Gm18 level, in turn, reduced immunostimulation properties of bacterial tRNAs, which is concordant with the previous observation that Gm18 is a suppressor of Toll-like receptor 7 (TLR7)-mediated interferon release. This documents an effect of stress induced modulation of tRNA modification that acts outside protein translation.
Substitution of the queuine nucleobase precursor preQ1 by an azide-containing derivative (azido-propyl-preQ1) led to incorporation of this clickable chemical entity into tRNA via transglycosylation in vitro as well as in vivo in Escherichia coli, Schizosaccharomyces pombe and human cells. The resulting semi-synthetic RNA modification, here termed Q-L1, was present in tRNAs on actively translating ribosomes, indicating functional integration into aminoacylation and recruitment to the ribosome. The azide moiety of Q-L1 facilitates analytics via click conjugation of a fluorescent dye, or of biotin for affinity purification. Combining the latter with RNAseq showed that TGT maintained its native tRNA substrate specificity in S. pombe cells. The semi-synthetic tRNA modification Q-L1 was also functional in tRNA maturation, in effectively replacing the natural queuosine in its stimulation of further modification of tRNAAsp with 5-methylcytosine at position 38 by the tRNA methyltransferase Dnmt2 in S. pombe. This is the first demonstrated in vivo integration of a synthetic moiety into an RNA modification circuit, where one RNA modification stimulates another. In summary, the scarcity of queuosinylation sites in cellular RNA, makes our synthetic q/Q system a ‘minimally invasive’ system for placement of a non-natural, clickable nucleobase within the total cellular RNA.
The fields of RNA modification and RNA damage both exhibit a plethora of non‐canonical nucleoside structures. While RNA modifications have evolved to improve RNA function, the term RNA damage implies detrimental effects. Based on stable isotope labelling and mass spectrometry, we report the identification and characterisation of 2‐methylthio‐1,N6‐ethenoadenosine (ms2ϵA), which is related to 1,N6‐ethenoadenine, a lesion resulting from exposure of nucleic acids to alkylating chemicals in vivo. In contrast, a sophisticated isoprene labelling scheme revealed that ms2ϵA biogenesis involves cleavage of a prenyl moiety in the known transfer RNA (tRNA) modification 2‐methylthio‐N6‐isopentenyladenosine (ms2i6A). The relative abundance of ms2ϵA in tRNAs from translating ribosomes suggests reduced function in comparison to its parent RNA modification, establishing the nature of the new structure in a newly perceived overlap of the two previously separate fields, namely an RNA modification damage.
Die Bereiche RNA‐Modifikation und RNA‐Schaden weisen beide eine Vielzahl nicht‐kanonischer Nukleosidstrukturen auf. Während sich RNA‐Modifikationen zur Verbesserung der RNA‐Funktion entwickelt haben, impliziert die Bezeichnung RNA‐Schaden negative Auswirkungen. Auf Grundlage der Markierung mit stabilen Isotopen und Massenspektrometrie berichten wir von der Identifizierung und Charakterisierung von 2‐Methylthio‐1,N6‐ethenoadenosin (ms2ϵA), welches mit 1,N6‐Ethenoadenin, einer Läsion, die durch Exposition von Nukleinsäuren gegenüber alkylierenden Chemikalien in vivo entsteht, verwandt ist. Im Gegensatz dazu zeigte ein ausgefeiltes Konzept zur Isopren‐Markierung, dass die Biogenese von ms2ϵA die Spaltung eines Prenylrests in der bekannten transfer‐RNA (tRNA)‐Modifikation 2‐Methylthio‐N6‐isopentenyladenosin (ms2i6A) beinhaltet. Die relative Häufigkeit von ms2ϵA in tRNAs von translatierenden Ribosomen lässt eine verminderte Funktionalität im Vergleich zur ursprünglichen RNA‐Modifikation vermuten, wodurch die Natur der neuen Struktur in einer neu wahrgenommenen Überschneidung der beiden zuvor getrennten Bereiche, nämlich ein RNA‐Modifikationsschaden, begründet wird.
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