The role of RNA modifications in the regulation of tRNA cleavage

Lyons, Shawn M.; Fay, Marta M.; Ivanov, Pavel

doi:10.1002/1873-3468.13205

Cited by 115 publications

(103 citation statements)

References 148 publications

(184 reference statements)

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“…Although post-transcriptional tRNA modifications have hindered efforts to sequence tRNAs, they are a universal and fundamental aspect of tRNA biology (Rojas-Benitez et al 2015;Lyons et al 2018).…”

Section: Introductionmentioning

confidence: 99%

“…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%

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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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Section: Introductionmentioning

confidence: 99%

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

View full text Add to dashboard Cite

show abstract

“…16 During this process, the 5' leader and 3' trailer in the pre-tRNA are removed by ribonuclease P (RNase P) and RNase Z respectively. 20 Furthermore, the complete maturation of certain tRNAs needs the removal of introns between positions 37 and 38. 20 Furthermore, the complete maturation of certain tRNAs needs the removal of introns between positions 37 and 38.…”

Section: Structure Of Trnasmentioning

confidence: 99%

“…[17][18][19] Subsequently, a CCA sequence associated with aminoacylation will be added to the 3' end by tRNA nucleotidyltransferase 1 (TRNT1). 20 Furthermore, the complete maturation of certain tRNAs needs the removal of introns between positions 37 and 38. In mammalian cells, an endonuclease complex that consists of homologs of the yeast tRNA endonuclease subunits is responsible for the cleavage reactions, followed by exon binding catalysed by a tRNA ligase complex containing the HSPC117 subunit.…”

Section: Structure Of Trnasmentioning

confidence: 99%

The tRNA‐associated dysregulation in immune responses and immune diseases

et al. 2019

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Transfer RNA (tRNA), often considered as a housekeeping molecule, mainly participates in protein translation by transporting amino acids to the ribosome. Nevertheless, accumulating evidence has shown that tRNAs are closely related to various physiological and pathological processes. The proper functioning of the immune system is the key to human health. The aim of this review is to investigate the relationships between tRNAs and the immune system. We detail the biogenesis and structure of tRNAs and summarize the pathogen tRNA-mediated infection and host responses. In addition, we address recent advances in different aspects of tRNA-associated dysregulation in immune responses and immune diseases, such as tRNA molecules, tRNA modifications, tRNA derivatives and tRNA aminoacylation. Therefore, tRNAs play an important role in immune regulation. Although our knowledge of tRNAs in the context of immunity remains, for the most part, unknown, this field deserves in-depth research to provide new ideas for the treatment of immune diseases. K E Y W O R D Sbiogenesis, immune diseases, immune responses, tRNA F I G U R E 1 The biogenesis and structure of tRNAs. tRNA: Transfer RNA; Pol III: RNA polymerase III; pre-tRNA: Precursor tRNA; mRNA: Messenger RNA; aaRS: Aminoacyl-tRNA synthetase; tsRNA: tRNA-derived small RNA; tRF: tRNA-derived fragment; tiRNA: tRNA-derived stress-induced RNA

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“…AQRNA-seq also captures the dynamics of tRNA fragmentation and degradation, as occurs in tRNA maturation and quality control, [32][33][34] small RNA regulation of gene expression, and toxin-antitoxin systems. [35][36][37][38][39][40] It is difficult to differentiate 5'-degradation of full-length tRNA from polymerase fall-off as both generate a fragment that aligns at the 3' end of tRNA ("type 2" reads, Fig. 4b).…”

Section: Starvation-induced Persistence Remodels the Trna Landscape Imentioning

confidence: 99%

Sequencing-based quantitative mapping of the cellular small RNA landscape

Yim

Huber

et al. 2019

Preprint

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Current next-generation RNA sequencing methods cannot provide accurate quantification of the population of small RNAs within a sample due to strong sequence-dependent biases in capture, ligation, and amplification during library preparation. We report the development of an RNA sequencing method -AQRNA-seq -that minimizes biases and enables absolute quantification of all small RNA species in a sample mixture. Validation of AQRNA-seq library preparation and data mining algorithms using a 963-member microRNA reference library, RNA oligonucleotide standards of varying lengths, and northern blots demonstrated a direct, linear correlation between sequencing read count and RNA abundance. Application of AQRNA-seq to bacterial tRNA pools, a traditionally hard-to-sequence class of RNAs, revealed 80-fold variation in tRNA isoacceptor copy numbers, patterns of site-specific tRNA fragmentation caused by stress, and quantitative maps of ribonucleoside modifications, all in a single AQRNA-seq experiment.AQRNA-seq thus provides a means to quantitatively map the small RNA landscape in cells and tissues.Next generation RNA sequencing platforms (RNA-seq) have advanced functional genomics by facilitating discoveries of new RNA species, gene annotations, and quantitative analysis of gene expression. 1, 2 RNA-seq has played a central role in unravelling the complexity of RNA function by facilitating quantitative profiling of the transcriptome as a function of cell state.Although a variety of RNA-seq methods provide precise and accurate analysis of changes in transcript abundance between samples or conditions, their major drawback is the inability to accurately quantify small RNA species within a sample. This is partly rooted in biased ligation of sequencing linkers to the 3'-and 5'-ends of RNAs, which varies by 10 3 -fold depending upon on the identity of the terminal nucleotides 3-7 and causes 10 6 -fold artifacts in sequencing read counts. 5,8,9 Highly structured and heavily modified RNA molecules, such as tRNAs, further challenge the quantitative accuracy of RNA-seq. 10, 11 These structural features cause polymerase fall-off during cDNA synthesis, which prevents detection of the transcripts.A variety of specialized RNA-seq methods, many limited specifically to either miRNA or tRNA only, 12-14 attempt to minimize ligation bias using linkers with randomized terminal nucleotides and molecular crowding agents to enhance ligation 4 and to reduce polymerase fall-off during reverse transcription (RT) with two-step ligation 8 and removal of some methyl modifications with AlkB. [15][16][17] Although polymerase fall-off effects are minimized, residual ligation biases persist and lead to "jackpot" sequences. 8 For example, the template-switching reverse transcriptase TGIRT used in DM-tRNA-seq 16 is biased by the identity of the overhanging nucleotide in the adapter strand. 18 The problem with existing RNA-seq techniques is that none have been systematically engineered to optimize ligation and amplification efficiencies or validated for quanti...

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The role of RNA modifications in the regulation of tRNA cleavage

Cited by 115 publications

References 148 publications

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

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

The tRNA‐associated dysregulation in immune responses and immune diseases

Sequencing-based quantitative mapping of the cellular small RNA landscape

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