Absolute Quantification of Noncoding RNA by Microscale Thermophoresis

Jacob, Dominik; Thüring, Kathrin; Galliot, Aurellia; Marchand, Virginie; Galvanin, Adeline; Ciftci, Akif; Scharmann, Karin; Stöck, Michael; Roignant, Jean‐Yves; Leidel, Sebastian A.; Motorin, Yuri; Schaffrath, Raffael; Klassen, Roland; Helm, Mark

doi:10.1002/anie.201814377

Cited by 30 publications

(22 citation statements)

References 34 publications

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“…Hence, the actual copy number of individual tRNAs or specific tsRNAs in any cell type under specific growth or stress conditions remains largely unknown. Microscale thermophoresis has been recently used to measure copy numbers of endogenous tRNAs [54]. As for tsRNAs, copy number estimates for particular tsRNA species have been attempted based on textbook calculations (for instance, in supplementary data in [20]), but these estimates remain experimentally unproven.…”

Section: Identification Of Proteins Associating With Endogenous 5ʹ Tsmentioning

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: Identification Of Proteins Associating With Endogenous 5ʹ Tsmentioning

confidence: 99%

Production and purification of endogenously modified tRNA-derived small RNAs

et al. 2020

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“…"Jackpot" tRNAs with extremely and presumably artefactually high read abundance have been reported in prior tRNA-seq results (Pang et al 2014;Jacob et al 2019), and the original YAMAT-seq paper reported a single tRNA-LysCTT gene having approximately 10-fold higher expression than the next most highly expressed gene (Shigematsu et al, 2017).…”

Section: The Promise and Pitfalls Of Quantifying Trna Expression Usinmentioning

confidence: 89%

Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification

Warren

Salinas‐Giegé

Hummel

et al. 2019

Preprint

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Differences in tRNA expression have been implicated in a remarkable number of biological processes.There is growing evidence that tRNA genes can play dramatically different roles depending on both expression and post-transcriptional modification, yet sequencing tRNAs to measure abundance and detect modifications remains challenging. Their secondary structure and extensive post-transcriptional modifications interfere with RNA-seq library preparation methods and have limited the utility of highthroughput sequencing technologies. Here, we combine two modifications to standard RNA-seq methods by treating with the demethylating enzyme AlkB and ligating with tRNA-specific adapters in order to sequence tRNAs from four species of flowering plants, a group that has been shown to have some of the most extensive rates of post-transcriptional tRNA modifications. This protocol has the advantage of detecting full-length tRNAs and sequence variants that can be used to infer many post-transcriptional modifications. We used the resulting data to produce a modification index of almost all unique reference tRNAs in Arabidopsis thaliana, which exhibited many anciently conserved similarities with humans but also positions that appear to be "hot spots" for modifications in angiosperm tRNAs. We also found evidence based on northern blot analysis and droplet digital PCR that, even after demethylation treatment, tRNA-seq can produce highly biased estimates of absolute expression levels most likely due to biased reverse transcription. Nevertheless, the generation of full-length tRNA sequences with modification data is still promising for assessing differences in relative tRNA expression across treatments, tissues or subcellular fractions and help elucidate the functional roles of tRNA modifications.

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“…In cases where modifications outside the ASL are lost, tRNA destabilization may account for the negative phenotype ( 74 ). However, when distinct ASL modifications are lost, tRNA remains stable but functionally impaired ( 2 , 15 , 77 ). This is well characterized for combined loss of mcm 5 U34 and s 2 U34, resulting in translational slowdown of the ribosome at CAA and AAA codons read by tRNA Gln UUG and tRNA Lys UUU , respectively.…”

Section: Discussionmentioning

confidence: 99%

Misactivation of multiple starvation responses in yeast by loss of tRNA modifications

Bruch

Laguna

Butter

et al. 2020

Nucleic Acids Research

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Previously, combined loss of different anticodon loop modifications was shown to impair the function of distinct tRNAs in Saccharomyces cerevisiae. Surprisingly, each scenario resulted in shared cellular phenotypes, the basis of which is unclear. Since loss of tRNA modification may evoke transcriptional responses, we characterized global transcription patterns of modification mutants with defects in either tRNAGlnUUG or tRNALysUUU function. We observe that the mutants share inappropriate induction of multiple starvation responses in exponential growth phase, including derepression of glucose and nitrogen catabolite-repressed genes. In addition, autophagy is prematurely and inadequately activated in the mutants. We further demonstrate that improper induction of individual starvation genes as well as the propensity of the tRNA modification mutants to form protein aggregates are diminished upon overexpression of tRNAGlnUUG or tRNALysUUU, the tRNA species that lack the modifications of interest. Hence, our data suggest that global alterations in mRNA translation and proteostasis account for the transcriptional stress signatures that are commonly triggered by loss of anticodon modifications in different tRNAs.

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Absolute Quantification of Noncoding RNA by Microscale Thermophoresis

Cited by 30 publications

References 34 publications

Production and purification of endogenously modified tRNA-derived small RNAs

Production and purification of endogenously modified tRNA-derived small RNAs

Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification

Misactivation of multiple starvation responses in yeast by loss of tRNA modifications

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