Time-resolved NMR monitoring of tRNA maturation

Barraud, Pierre; Gato, Alexandre; Heiss, MM; Catala, Marjorie; Kellner, Stefanie; Tisné, Carine

doi:10.1038/s41467-019-11356-w

Cited by 67 publications

(86 citation statements)

References 69 publications

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“…LC-MS/MS analyses can reproducibly quantify individual RNA modifications in purified tRNAs as shown by analysing six LC-MS/MS measurements on commercially available tRNA-Phe from Saccharomyces cerevisiae (supplementary Figure 3A and B confirming the analysis of S.c. tRNA-Phe published in [53]). 5ʹ tsRNA-Gly GCC/CCC and 5ʹ tsRNA-Glu CUC/UUC along with their remaining parental full-length tRNAs were purified from biological triplicate experiments after either iAs exposure or ecANG expression followed by LC-MS/MS analyses (Fig.…”

Section: Determining the Modification Status Of Purified Tsrnasmentioning

confidence: 99%

Production and purification of endogenously modified tRNA-derived small RNAs

et al. 2020

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2020): Production and purification of endogenously modified tRNA-derived small RNAs, RNA Biology, ABSTRACT During particular stress conditions, transfer RNAs (tRNAs) become substrates of stress-induced endonucleases, resulting in the production of distinct tRNA-derived small RNAs (tsRNAs). These small RNAs have been implicated in a wide range of biological processes, but how isoacceptor and even isodecoder-specific tsRNAs act at the molecular level is still poorly understood. Importantly, stress-induced tRNA cleavage affects only a few tRNAs of a given isoacceptor or isodecoder, raising the question as to how such limited molecule numbers could exert measurable biological impact. While the molecular function of individual tsRNAs is likely mediated through association with other molecules, addressing the interactome of specific tsRNAs has only been attempted by using synthetic RNA sequences. Since tRNAs carry post-transcriptional modifications, tsRNAs are likely modified but the extent of their modifications remains largely unknown. Here, we developed a biochemical framework for the production and purification of specific tsRNAs using human cells. Preparative scale purification of tsRNAs from biological sources should facilitate experimentally addressing as to how exactly these small RNAs mediate the multitude of reported molecular functions. ARTICLE HISTORY

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Section: Determining the Modification Status Of Purified Tsrnasmentioning

confidence: 99%

Production and purification of endogenously modified tRNA-derived small RNAs

et al. 2020

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“…U55 to Ψ55 as observed in yeast 22 . In fact, we observe 1.5 fold more Ψ in the early lifetime of tRNA Phe GAA as is expected from mature tRNA Phe GAA (Figure 1).…”

Section: Temporal Placement Of Modified Nucleosides In Rnamentioning

confidence: 67%

“…By NMR spectroscopy in combination with stable isotope labeling, Barraud et al recently observed an inhibition of m 22 G formation by m 2 G 22 . In our hands, m 22 G is placed into tRNA Phe as fast as is m 2 G, but as both modifications are incorporated slowly it is possible that m 22 G is placed in a non-m 2 G modified sub-population. This question might be approached by combining NAIL with oligonucleotide MS.…”

Section: Discussionmentioning

confidence: 95%

“…tRNA Phe is heavily post-transcriptionally modified and in addition one of the best studied RNAs [19][20][21][22] .…”

Section: Absolute Quantification Of Human Trna Phe Modificationsmentioning

confidence: 99%

“…For example, tRNA Phe is heavily posttranscriptionally modified and in addition one of the best studied RNAs [19][20][21] . By using stable isotope labeled tRNA Phe substrate and cellular extracts, the modification dynamics and hierarchy was recently solved in S. cerevisiae using NMR spectroscopy 22 . Under the influence of chemical stress, S. cerevisiae was reported to adapt its abundance of tRNA modifications and thus influence its translation and the term stress induced tRNA reprogramming was coined 11,23 .…”

Section: Introductionmentioning

confidence: 99%

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Cell culture NAIL-MS allows insight into human RNA modification dynamicsin vivo

Heiss

Hagelskamp

Kellner

2020

Preprint

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In the last years, studies about the dynamics of RNA modifications are among the most controversially discussed. As the main reason, we have identified the unavailability of a technique which allows to follow the temporal dynamics of RNA transcripts in human cell culture.Here, we present a NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) scheme for efficient stable isotope labeling in both RNA and DNA (>95% within 7 days) in common human cell lines and growth media. Validation experiments reveal that the labeling procedure itself does neither interfere with the isotope dilution MS quantification nor with RNA modification density. We design pulse chase NAIL-MS experiments and apply the new tool to study the temporal placement of modified nucleosides in e.g. tRNA Phe and 18S rRNA. In existing RNAs, we observe a constant loss of modified nucleosides over time which is masked by a post-transcriptional methylation mechanism and thus not detectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing transcripts.Overall, we present a fast and reliable stable isotope labeling strategy which allows a more detailed study of RNA modification dynamics in human cell culture. With cell culture NAIL-MS it is finally possible to study the speed of both modification and demethylation reactions inside human cells. Thus it will be possible to study the impact of external stimuli and stress on human RNA modification kinetics and processing of mature RNA.

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Functional interplay within the epitranscriptome: Reality or fiction?

2021

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RNA modifications have recently emerged as an important regulatory layer of gene expression. The most prevalent and reversible modification on messenger RNA (mRNA), N6‐methyladenosine, regulates most steps of RNA metabolism and its dysregulation has been associated with numerous diseases. Other modifications such as 5‐methylcytosine and N1‐methyladenosine have also been detected on mRNA but their abundance is lower and still debated. Adenosine to inosine RNA editing is widespread on coding and non‐coding RNA and can alter mRNA decoding as well as protect against autoimmune diseases. 2′‐O‐methylation of the ribose and pseudouridine are widespread on ribosomal and transfer RNA and contribute to proper RNA folding and stability. While the understanding of the individual role of RNA modifications has now reached an unprecedented stage, still little is known about their interplay in the control of gene expression. In this review we discuss the examples where such interplay has been observed and speculate that with the progress of mapping technologies more of those will rapidly accumulate.

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Time-resolved NMR monitoring of tRNA maturation

Cited by 67 publications

References 69 publications

Production and purification of endogenously modified tRNA-derived small RNAs

Production and purification of endogenously modified tRNA-derived small RNAs

Cell culture NAIL-MS allows insight into human RNA modification dynamicsin vivo

Functional interplay within the epitranscriptome: Reality or fiction?

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