The Versatile Roles of the tRNA Epitranscriptome during Cellular Responses to Toxic Exposures and Environmental Stress

Huber, Sabrina M.; Leonardi, Andrea; Dedon, Peter C.; Begley, Thomas J.

doi:10.3390/toxics7010017

Cited by 69 publications

(71 citation statements)

References 114 publications

(151 reference statements)

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“…The tRNA methyltransferase (AT5G15810) was shown to cause stress-related N2, N2dimethylguanosine (m 2 2 G) modification in tRNAs of A. thaliana [110]. Usually, tRNA nucleotide modifications occur within tRNAs during their maturation and processing and these modifications are biomarkers of specific stresses and were observed to be induced in response to oxidizing agents [111]. It is also known that stress-induced epitranscriptomic changes regulate tRNA stability, translation initiation, and microRNA-based regulation of transcripts [111].…”

Section: Analysis Of Mirnas and Their Putative Targetsmentioning

confidence: 99%

Identification of small RNAs during cold acclimation in Arabidopsis thaliana

et al. 2020

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Background: Cold stress causes dynamic changes in gene expression that are partially caused by small non-coding RNAs since they regulate protein coding transcripts and act in epigenetic gene silencing pathways. Thus, a detailed analysis of transcriptional changes of small RNAs (sRNAs) belonging to all known sRNA classes such as microRNAs (miRNA) and small interfering RNA (siRNAs) in response to cold contributes to an understanding of cold-related transcriptome changes. Result: We subjected A. thaliana plants to cold acclimation conditions (4°C) and analyzed the sRNA transcriptomes after 3 h, 6 h and 2 d. We found 93 cold responsive differentially expressed miRNAs and only 14 of these were previously shown to be cold responsive. We performed miRNA target prediction for all differentially expressed miRNAs and a GO analysis revealed the overrepresentation of miRNA-targeted transcripts that code for proteins acting in transcriptional regulation. We also identified a large number of differentially expressed cisand trans-nat-siRNAs, as well as sRNAs that are derived from long non-coding RNAs. By combining the results of sRNA and mRNA profiling with miRNA target predictions and publicly available information on transcription factors, we reconstructed a cold-specific, miRNA and transcription factor dependent gene regulatory network. We verified the validity of links in the network by testing its ability to predict target gene expression under cold acclimation. Conclusion: In A. thaliana, miRNAs and sRNAs derived from cisand trans-NAT gene pairs and sRNAs derived from lncRNAs play an important role in regulating gene expression in cold acclimation conditions. This study provides a fundamental database to deepen our knowledge and understanding of regulatory networks in cold acclimation.

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Section: Analysis Of Mirnas and Their Putative Targetsmentioning

confidence: 99%

Identification of small RNAs during cold acclimation in Arabidopsis thaliana

et al. 2020

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“…Base modifications are involved in tRNA folding and stability (Sampson and Uhlenbeck 1988;Vermeulen et al 2005;Phizicky and Alfonzo 2010), translational accuracy and reading-frame maintenance (Urbonavicius et al 2001;Hou et al 2015), and tRNA fragment generation (Lyons et al 2018;Huber et al 2019). Not surprisingly given these fundamental roles, they have been implicated in numerous diseases (Phizicky and Hopper 2015), and there has been a long historical interest to identify, map, and quantify these modifications (Kuchino et al 1987).…”

Section: Introductionmentioning

confidence: 99%

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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“…While CM standards resemble the complexity of biological samples, the RNA isolates often contain impurities that may impair MS analysis. Transfer RNA (tRNA) contains on average 13 ribonucleoside modifications per molecule, although the exact number and type of modifications vary between different organisms and tissue types or due to growth conditions and/or presence of stress 3,4,8,30,36 . To determine if our method can robustly identify and quantitatively characterize relative (Figure 2A, Supplementary Figure S6).…”

Section: C18 Uplc-ms Provides Robust Identification Of Ribonucleosidementioning

confidence: 99%

“…During the past decade, research into post-transcriptional nucleoside modifications -in particular those of messenger RNA and transfer RNA -has experienced a renaissance as their multifaceted roles, ranging from translational control 1 via infection 2 and stress response 3,4 to development and aging 5,6 , are slowly beginning to unfold. This can be credited to methods and instrument development 2 in two key areas; (I) translation studies using ribosome profiling combined with next-generation sequencing 7,8 , which enables detailed insights into the translational state of cells, and (II) identification and quantitative characterization of modified ribonucleosides by liquid chromatography mass spectrometry (LC-MS).…”

Section: Introductionmentioning

confidence: 99%

Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS

Gregorová

Sipari

Sarin

2020

Preprint

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Post-transcriptional RNA modifications play an important role in cellular metabolism with homeostatic disturbances manifesting as a wide repertoire of phenotypes, reduced stress tolerance and translational perturbation, developmental defects, and diseases, such as type II diabetes, leukemia and carcinomas. Hence, there has been an intense effort to develop various methods for investigating RNA modifications and their roles in various organisms, including sequencing-based approaches and, more frequently, liquid chromatography mass spectrometry (LC-MS)-based methods. Although LC-MS offers numerous advantages, such as being highly sensitive and quantitative over a broad detection range, some stationary phase chemistries struggle to resolve positional isomers. Furthermore, the demand for detailed analyses of complex biological samples often necessitates long separation times, hampering sample-to-sample turnover and making multisample analyses time consuming. To overcome this limitation, we have developed an ultra-performance LC-MS (UPLC-MS) method that uses an octadecyl carbon chain (C18)-bonded silica matrix for the efficient separation of 50 modified ribonucleosides, including positional isomers, in a single 9 min sample-to-sample run. To validate the performance and versatility of our method, we analyzed tRNA modification patterns of representative microorganisms from each kingdom of life, namely Archaea (Methanosarcina acetivorans), Bacteria (Pseudomonas syringae) and Eukarya (Saccharomyces cerevisiae). Additionally, our method is flexible and readily applicable for detection and relative quantification using stable isotope labelling and targeted approaches like multiple reaction monitoring (MRM). In conclusion, this method represents a fast and robust tool for broad-range exploration and quantification of ribonucleosides, facilitating future homeostasis studies of RNA modification in complex biological samples.

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The Versatile Roles of the tRNA Epitranscriptome during Cellular Responses to Toxic Exposures and Environmental Stress

Cited by 69 publications

References 114 publications

Identification of small RNAs during cold acclimation in Arabidopsis thaliana

Identification of small RNAs during cold acclimation in Arabidopsis thaliana

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

Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS

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