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Modifications in the T arm of tRNA globally determine tRNA function and cellular fitness

ABSTRACT: All elongator tRNAs harbor 5-methyluridine at position 54 and pseudouridine at position 55 in the T arm, which are generated by the enzymes TrmA and TruB, respectively. Escherichia coli TrmA and TruB have both been shown to act as tRNA chaperones, and strains lacking trmA or truB are outcompeted by wildtype. Here, we investigate how TrmA and TruB contribute to cellular fitness. Deletion of trmA and truB in E. coli causes a global decrease in aminoacylation and alters other tRNA modification such as acp3U47 and 4-thiouridine. Whereas global protein synthesis is not significantly changed in ΔtrmA and ΔtruB, the expression of many specific proteins is altered at the translational level. In conclusion, we demonstrate that universal modifications of the tRNA T arm are critical for global tRNA function by enhancing other tRNA modifications, tRNA folding, tRNA aminoacylation, and translation of specific genes thereby improving cellular fitness and explaining their conservation.

ORGANISM(S): Escherichia coli

PROVIDER: GSE237609 | GEO | 2024/06/05

REPOSITORIES: GEO

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Modifications in the T arm of tRNA globally determine tRNA function and cellular fitness

Project description:Abstract: tRNAs are highly modified in the elbow region and harbor 5-methyluridine at position 54 and pseudouridine at position 55 in the T arm, which are generated by the enzymes TrmA and TruB, respectively. Although all elongator tRNAs contain these modifications across all domains of life, the cellular relevance of these modifications and their corresponding modifying enzymes remains elusive. In addition to modifying every tRNA, Escherichia coli TrmA and TruB have both been shown to fold tRNA independently of its modification activity acting as tRNA chaperones, and strains lacking trmA or truB are outcompeted by wildtype. To identify how TrmA and TruB contribute to cellular fitness, we have systematically assessed the effects of deleting trmA and/or trmB in E. coli. Since tRNA folding is a pre-requisite for tRNA aminoacylation, we determined cellular aminoacylation levels revealing a global decrease in aminoacylation for all tRNAs in ΔtrmA and ΔtruB. Moreover, the absence of 5-methyluridine 54 or pseudouridine 55 alters tRNA modification at other positions: whereas acp3U47 is decreased, thiouridine levels are increased. Understanding the importance of TrmA and TruB for tRNA aminoacylation and modification, we then analyzed how these global tRNA changes in ΔtrmA and ΔtruB strains affect translation. Whereas global protein synthesis is not significantly changed in ΔtrmA and ΔtruB, the abundances of many specific proteins are altered, and transcriptomics experiments suggest that the dysregulation of many proteins is controlled at the translational level. In conclusion, we demonstrate that universally conserved modifications of the tRNA elbow are critical for global tRNA function by enhancing other tRNA modifications, tRNA folding, tRNA aminoacylation and translation of specific genes thereby contributing to cellular fitness.

2023-05-31 | GSE233667 | GEO

Conserved 5-methyluridine tRNA modification modulates ribosome translocation

Project description:While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA inE. coli, Trm2 inS. cerevisiae) exhibit altered tRNA modifications patterns. Furthermore, m5U54 deficient tRNAs are desensitized to small molecules that prevent translocationin vitro.This finding is consistent with our observations that, relative to wild-type cells,trm2Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.

2024-08-08 | GSE273506 | GEO

Suppressor mutant screening lacking growth-essential tRNA modification in bacteria

Project description:Post-transcriptional modifications are important for transfer RNAs (tRNAs) to be efficient and accurate in translation on the ribosome. The m1G37 modification on a subset of tRNAs in bacteria are generated by a conserved methyltransferase TrmD and is essential for bacterial growth. Previous studies showed that m1G37 has an important role in preventing translational frameshifting and also that this modification is coupled with aminoacylation of tRNAs for proline. Here we performed suppressor screening to isolate a mutant E. coli cell that lacks TrmD but is viable, and the whole-genome sequencing revealed several mutations on prolyl-tRNA synthetase (ProRS) gene conferring cell viability in the absence of TrmD. Biochemical assays confirmed uncoupling of m1G37 modification and aminoacylation, and cell-based assays uncovered the critical role of m1G37 in supporting Wobble decoding.

2022-09-24 | E-MTAB-12178 | biostudies-arrayexpress

tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase

Project description:Heterozygous mutations in six tRNA synthetase genes cause Charcot-Marie-Tooth (CMT) peripheral neuropathy. CMT-mutant glycyl- or tyrosyl-tRNA synthetases inhibit global protein synthesis by an unknown mechanism, independent of aminoacylation activity. We report that tRNAGly overexpression rescues protein synthesis and peripheral neuropathy phenotypes in Drosophila and mouse models of CMT caused by glycyl-tRNA synthetase (GlyRS) mutations (CMT2D). Kinetic experiments revealed that CMT-mutant GlyRS bind tRNAGly, but display markedly slow release rates. This tRNAGly sequestration may deplete the cellular tRNAGly pool, leading to insufficient glycyl-tRNAGly supply to the ribosome and translation deficit.

2021-09-07 | GSE160584 | GEO

Lack of TRMT2A, a tRNA methyltransferase, induces the generation of tRNA-derived small RNAs due to m5U54 tRNA hypomodification [Seq]

Project description:Transfer RNA (tRNA) molecules contain a variety of post-transcriptional modifications which are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation, to gene expression control and cellular stress response. Recent evidence show that tsRNAs are also modified, however the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. This modification is catalyzed by the tRNA methyltransferase TRMT2A, but its biological role remains largely unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification, resulting in angiogenin (ANG) dependent tsRNA formation. More specifically, m5U54 hypomodification is followed by ANG overexpression and tRNA cleavage near the anticodon, resulting in accumulation of 5’tRNA-derived stress-induced RNAs (5’tiRNAs), in particular 5’tiRNA-GlyGCC and 5’tiRNA-GluCTC. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently m5U54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tsRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tsRNA formation in mammalian cells. These results establish a link between tRNA demethylation and tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.

2021-04-20 | GSE164405 | GEO

Lack of TRMT2A, a tRNA methyltransferase, induces the generation of tRNA-derived small RNAs due to m5U54 tRNA hypomodification [Array]

2021-04-20 | GSE164168 | GEO

Profiling tRNA modifications in Mycobacterium tuberculosis

Project description:Diverse chemical modifications fine-tune the function and metabolism of tRNA. Although tRNA modification is universal in all kingdoms of life, profiles of modifications, their functions, and physiological roles have not been elucidated in most organisms including the human pathogen, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To identify physiologically important modifications, we surveyed the tRNA of Mtb, using tRNA sequencing (tRNA-seq). Reverse transcription-derived error signatures in tRNA-seq predicted the sites and presence of 9 modifications. Several chemical treatments prior to tRNA-seq expanded the number of predictable modifications. Deletion of Mtb genes encoding two modifying enzymes, TruB and MnmA, eliminated their respective tRNA modifications, validating the presence of modified sites in tRNA species.

2023-08-11 | GSE240200 | GEO

McFarland2020 - Synthetase sequestration Model:a combined translation and tRNA charging cycle

Project description:The Synthetase Sequestration Model (SSM) is a simplified translation model that considers explicitly two main steps in the process of tRNA aminoacylation: first, the tRNA is bound by the aminoacyl tRNA synthetase, and in a second step, the amino acid is attached to the tRNA. The tRNA then participates in the translation reaction, becoming deacylated as a result. The tRNA exists in states bound, charged and uncharged. In the bound state, the tRNA is bound to the synthetase but uncharged, i.e., the tRNA is sequestered by the synthetase. The model predicts how the balance between the three different tRNA states (empty, bound and charged) changes depending on aminoacyl tRNA synthetase availability.

2020-02-05 | MODEL2001080005 | BioModels

Tarsl2 is evolved not for tRNA charging in vivo

Project description:As a newly evolved duplicated gene of TARS1 (encoding cytoplasmic threonyl-tRNA synthetase), TARSL2 (TARS3) encodes a protein highly similar with TARS1 with only obvious difference in N-terminus. Albeit with tRNA charging activity in vitro and incorporated into the multiple tRNA synthetase complex (MSC), whether TARSL2 is able to function as a tRNA synthetase to substitute TARS1 in vivo is unclear. In this work, we found that Tars1 was essential for embryonic development. In contrast, knockout of Tarsl2 was achievable. Loss of Tarsl2 had no effect on both abundance and charging levels of all tRNAThr isoacceptors, indicating Tarsl2 is not for tRNAThr metabolism. Furthermore, Tarsl2 deletion did not influence protein levels of other MSC components, MSC integrity and aminoacylation levels of non-cognate tRNAs by MSC components. However, a severe retardation of mice development was observed after 3 weeks accompanied with an elevated metabolism capacity and muscle development abnormity. We further constructed a Tarsl2-deleted zebrafish, which exhibited no change in the level and charging of tRNAThrs. Collectively, these data suggest that Tarsl2 is evolved not for canonical tRNAThr aminoacylation and cannot substitute Tars1 in vivo.

2023-05-31 | GSE218318 | GEO

Phenylalanine-tRNA aminoacylation is compromised by ALS/FTD-associated C9orf72 C4G2 repeat RNA

Project description:The expanded hexanucleotide GGGGCC repeat mutation in the C9orf72 gene is the main genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Under one disease mechanism, sense and antisense transcripts of the repeat are predicted to bind various RNA-binding proteins, compromise their function and cause cytotoxicity. Focusing on cytoplasmic interaction of the CCCCGG antisense repeat RNA this study identifies phenylalanine-tRNA synthetase (FARS) subunit alpha (FARSA) as the main interactor. The aminoacylation of tRNAPhe by FARS is inhibited by antisense RNA, leading to decreased levels of charged tRNAPhe. Remarkably, this is associated with global reduction of phenylalanine incorporation in the proteome and significant decrease in expression of phenylalanine-rich proteins in cellular models and patient tissues. In conclusion, this research reveals functional inhibition of FARSA in the presence of antisense RNA repeats. Compromised aminoacylation of tRNA could lead to impairments in protein synthesis and further contribute to C9orf72 mutation-associated pathology.

2023-09-18 | PXD042474 | Pride

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