tRNA Stability Research Articles

M5U54 tRNA Hypomodification by Lack of TRMT2A Drives the Generation of tRNA-Derived Small RNAs

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that 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 indicates 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. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification and tsRNA formation. More specifically, m5U54 hypomodification is followed by overexpression of the ribonuclease angiogenin (ANG) that cleaves tRNAs near the anticodon, resulting in accumulation of 5′tRNA-derived stress-induced RNAs (5′tiRNAs), namely 5′tiRNA-GlyGCC and 5′tiRNA-GluCTC, among others. 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 tiRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNA formation in mammalian cells. These results establish a link between tRNA hypomethylation and ANG-dependent tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.

Revisiting tRNA chaperones: New players in an ancient game.

tRNAs undergo an extensive maturation process including posttranscriptional modifications that influence secondary and tertiary interactions. Precursor and mature tRNAs lacking key modifications are often recognized as aberrant and subsequently targeted for decay, illustrating the importance of modifications in promoting structural integrity. tRNAs also rely on tRNA chaperones to promote the folding of misfolded substrates into functional conformations. The best characterized tRNA chaperone is the La protein, which interacts with nascent RNA polymerase III transcripts to promote folding and offers protection from exonucleases. More recently, certain tRNA modification enzymes have also been demonstrated to possess tRNA folding activity distinct from their catalytic activity, suggesting that they may act as tRNA chaperones. In this review, we will discuss pioneering studies relating posttranscriptional modification to tRNA stability and decay pathways, present recent advances into the mechanism by which the RNA chaperone La assists pre-tRNA maturation, and summarize emerging research directions aimed at characterizing modification enzymes as tRNA chaperones. Together, these findings shed light on the importance of tRNA folding and how tRNA chaperones, in particular, increase the fraction of nascent pre-tRNAs that adopt a folded, functional conformation.

Distinct evolutionary pathways for the synthesis and function of tRNA modifications.

Transfer ribonucleicacids (RNAs) (tRNAs) are essential adaptor molecules for translation. The functions and stability of tRNAs are modulated by their post-transcriptional modifications (tRNA modifications). Each domain of life has a specific set of modifications that include ones shared in multiple domains and ones specific to a domain. In some cases, different tRNA modifications across domains have similar functions to each other. Recent studies uncovered that distinct enzymes synthesize the same modification in different organisms, suggesting that such modifications are acquired through independent evolution. In this short review, I outline the mechanisms by which various modifications contribute to tRNA function, including modulation of decoding and tRNA stability, using recent findings. I also focus on modifications that are synthesized by distinct biosynthetic pathways.

Leber's Hereditary Optic Neuropathy: the roles of mitochondrial transfer RNA variants.

Leber’s Hereditary Optic Neuropathy (LHON) was a common maternally inherited disease causing severe and permanent visual loss which mostly affects males. Three primary mitochondrial DNA (mtDNA) mutations, ND1 3460G>A, ND4 11778G>A and ND6 14484T>C, which affect genes encoding respiratory chain complex I subunit, are responsible for >90% of LHON cases worldwide. Families with maternally transmitted LHON show incomplete penetrance with a male preponderance for visual loss, suggesting the involvement of secondary mtDNA variants and other modifying factors. In particular, variants in mitochondrial tRNA (mt-tRNA) are important risk factors for LHON. These variants decreased the tRNA stability, prevent tRNA aminoacylation, influence the post-transcriptionalmodification and affect tRNA maturation. Failure of mt-tRNA metabolism subsequently impairs protein synthesis and expression, folding, and function of oxidative phosphorylation (OXPHOS) enzymes, which aggravates mitochondrial dysfunction that is involved in the progression and pathogenesis of LHON. This review summarizes the recent advances in our understanding of mt-tRNA biology and function, as well as the reported LHON-related mt-tRNA second variants; it also discusses the molecular mechanism behind the involvement of these variants in LHON.

Posttranscriptional modifications in mitochondrial tRNA and its implication in mitochondrial translation and disease.

A fundamental aspect of mitochondria is that they possess DNA and protein translation machinery. Mitochondrial DNA encodes 22 tRNAs that translate mitochondrial mRNAs to 13 polypeptides of respiratory complexes. Various chemical modifications have been identified in mitochondrial tRNAs via complex enzymatic processes. A growing body of evidence has demonstrated that these modifications are essential for translation by regulating tRNA stability, structure and mRNA binding, and can be dynamically regulated by the metabolic environment. Importantly, the hypomodification of mitochondrial tRNA due to pathogenic mutations in mitochondrial tRNA genes or nuclear genes encoding modifying enzymes can result in life-threatening mitochondrial diseases in humans. Thus, the mitochondrial tRNA modification is a fundamental mechanism underlying the tight regulation of mitochondrial translation and is essential for life. In this review, we focus on recent findings on the physiological roles of 5-taurinomethyl modification (herein referred as taurine modification) in mitochondrial tRNAs. We summarize the findings in human patients and animal models with a deficiency of taurine modifications and provide pathogenic links to mitochondrial diseases. We anticipate that this review will help understand the complexity of mitochondrial biology and disease.

Archaeosine Modification of Archaeal tRNA: Role in Structural Stabilization.

Archaeosine (G+) is a structurally complex modified nucleoside found quasi-universally in the tRNA of Archaea and located at position 15 in the dihydrouridine loop, a site not modified in any tRNA outside the Archaea G+ is characterized by an unusual 7-deazaguanosine core structure with a formamidine group at the 7-position. The location of G+ at position 15, coupled with its novel molecular structure, led to a hypothesis that G+ stabilizes tRNA tertiary structure through several distinct mechanisms. To test whether G+ contributes to tRNA stability and define the biological role of G+, we investigated the consequences of introducing targeted mutations that disrupt the biosynthesis of G+ into the genome of the hyperthermophilic archaeon Thermococcus kodakarensis and the mesophilic archaeon Methanosarcina mazei, resulting in modification of the tRNA with the G+ precursor 7-cyano-7-deazaguansine (preQ0) (deletion of arcS) or no modification at position 15 (deletion of tgtA). Assays of tRNA stability from in vitro-prepared and enzymatically modified tRNA transcripts, as well as tRNA isolated from the T. kodakarensis mutant strains, demonstrate that G+ at position 15 imparts stability to tRNAs that varies depending on the overall modification state of the tRNA and the concentration of magnesium chloride and that when absent results in profound deficiencies in the thermophily of T. kodakarensisIMPORTANCE Archaeosine is ubiquitous in archaeal tRNA, where it is located at position 15. Based on its molecular structure, it was proposed to stabilize tRNA, and we show that loss of archaeosine in Thermococcus kodakarensis results in a strong temperature-sensitive phenotype, while there is no detectable phenotype when it is lost in Methanosarcina mazei Measurements of tRNA stability show that archaeosine stabilizes the tRNA structure but that this effect is much greater when it is present in otherwise unmodified tRNA transcripts than in the context of fully modified tRNA, suggesting that it may be especially important during the early stages of tRNA processing and maturation in thermophiles. Our results demonstrate how small changes in the stability of structural RNAs can be manifested in significant biological-fitness changes.

Biogenesis and functions of aminocarboxypropyluridine in tRNA

Transfer (t)RNAs contain a wide variety of post-transcriptional modifications, which play critical roles in tRNA stability and functions. 3-(3-amino-3-carboxypropyl)uridine (acp3U) is a highly conserved modification found in variable- and D-loops of tRNAs. Biogenesis and functions of acp3U have not been extensively investigated. Using a reverse-genetic approach supported by comparative genomics, we find here that the Escherichia coli yfiP gene, which we rename tapT (tRNA aminocarboxypropyltransferase), is responsible for acp3U formation in tRNA. Recombinant TapT synthesizes acp3U at position 47 of tRNAs in the presence of S-adenosylmethionine. Biochemical experiments reveal that acp3U47 confers thermal stability on tRNA. Curiously, the ΔtapT strain exhibits genome instability under continuous heat stress. We also find that the human homologs of tapT, DTWD1 and DTWD2, are responsible for acp3U formation at positions 20 and 20a of tRNAs, respectively. Double knockout cells of DTWD1 and DTWD2 exhibit growth retardation, indicating that acp3U is physiologically important in mammals.

IRNA-m2G: Identifying N2-methylguanosine Sites Based on Sequence-Derived Information

RNA N2-methylguanosine (m2G) is one kind of posttranscriptional modification and plays crucial roles in the control and stabilization of tRNA. However, our knowledge about the biological functions of m2G is still limited. The key step of revealing its new function is to recognize the m2G sites in the transcriptome. Since there is no effective method for detecting m2G sites, it is desirable to develop new methods to identify m2G sites. In this study, a computational predictor called iRNA-m2G was proposed to identify m2G sites in eukaryotic transcriptomes. In iRNA-m2G, the RNA sequences were encoded by using nucleotide chemical property and accumulated nucleotide frequency. iRNA-m2G was not only validated by the rigorous jackknife test on the benchmark dataset but also examined by performing cross-species validations. In addition, iRNA-m2G was also tested on an independent dataset. It was found that the accuracies obtained by iRNA-m2G were all quite promising in these tests, indicating that the proposed method could become a powerful tool for identifying m2G sites. Finally, a user-friendly web server for iRNA-m2G is freely accessible at http://lin-group.cn/server/iRNA-m2G.php.

NSUN2 introduces 5-methylcytosines in mammalian mitochondrial tRNAs.

Expression of human mitochondrial DNA is indispensable for proper function of the oxidative phosphorylation machinery. The mitochondrial genome encodes 22 tRNAs, 2 rRNAs and 11 mRNAs and their post-transcriptional modification constitutes one of the key regulatory steps during mitochondrial gene expression. Cytosine-5 methylation (m5C) has been detected in mitochondrial transcriptome, however its biogenesis has not been investigated in details. Mammalian NOP2/Sun RNA Methyltransferase Family Member 2 (NSUN2) has been characterized as an RNA methyltransferase introducing m5C in nuclear-encoded tRNAs, mRNAs and microRNAs and associated with cell proliferation and differentiation, with pathogenic variants in NSUN2 being linked to neurodevelopmental disorders. Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. Using three genetic models for NSUN2 inactivation—knockout mice, patient-derived fibroblasts and CRISPR/Cas9 knockout in human cells—we show that NSUN2 is necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs. Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. We discuss the importance of the newly discovered function of NSUN2 in the context of human disease.

Importance of the m1G37 modification and 32–38 pairing in tRNAPro(CCG) on decoding and tRNA stability

The ribosome is a complex and highly conserved molecular machine that is responsible for cellular protein synthesis (translation) in all three domains of life. During translation, the ribosome deco...

Structural studies on dihydrouridine synthase A (DusA) from Pseudomonas aeruginosa.

Dihydrouridination is one of the abundant modifications in tRNA editing. The presence of dihydrouridine is attributed to tRNA stability desired for the efficient gene translation process. The conversion of uridine to dihydrouridine is catalyzed by flavine containing enzyme called dihydrouridine synthase (Dus). We report first ever information about DusA enzyme from Pseudomonas aeruginosa in form of structural and functional studies. The gene coding for DusA from P. aeruginosa (PADusA) was cloned, expressed and purified, using recombinant DNA technology methods. Thermal and chemical stability of PADusA was determined with respect to temperature and urea-induced equilibrium unfolding experiments, with monitoring the change of ellipticity at 200-260 nm by Circular Dichroism (CD) spectroscopy. Unfolding studies revealed that PADusA has acquired a stable tertiary structure fold with a Tm value of 46.2 °C and Cm of 2.7 M for urea. The enzyme contains 43% α-helices and 16% β-strands. The three dimensional structure of PADusA was modeled using insilico methods. In order to understand the mechanism of substrate recognition and catalysis, tRNA and puromycin were docked on PADusA structure and their binding was analyzed. The structural features suggested that PADusA may also form a novel target for structure based drug design of antimicrobial agents.

The RNA degradosome promotes tRNA quality control through clearance of hypomodified tRNA

The factors and mechanisms that govern tRNA stability in bacteria are not well understood. Here, we investigated the influence of posttranscriptional modification of bacterial tRNAs (tRNA modification) on tRNA stability. We focused on ThiI-generated 4-thiouridine (s4U), a modification found in bacterial and archaeal tRNAs. Comprehensive quantification of Vibrio cholerae tRNAs revealed that the abundance of some tRNAs is decreased in a ΔthiI strain in a stationary phase-specific manner. Multiple mechanisms, including rapid degradation of a subset of hypomodified tRNAs, account for the reduced abundance of tRNAs in the absence of thiI Through transposon insertion sequencing, we identified additional tRNA modifications that promote tRNA stability and bacterial viability. Genetic analysis of suppressor mutants as well as biochemical analyses revealed that rapid degradation of hypomodified tRNA is mediated by the RNA degradosome. Elongation factor Tu seems to compete with the RNA degradosome, protecting aminoacyl tRNAs from decay. Together, our observations describe a previously unrecognized bacterial tRNA quality control system in which hypomodification sensitizes tRNAs to decay mediated by the RNA degradosome.

TRNA-Derived Small RNAs: Biogenesis, Modification, Function and Potential Impact on Human Disease Development.

Transfer RNAs (tRNAs) are abundant small non-coding RNAs that are crucially important for decoding genetic information. Besides fulfilling canonical roles as adaptor molecules during protein synthesis, tRNAs are also the source of a heterogeneous class of small RNAs, tRNA-derived small RNAs (tsRNAs). Occurrence and the relatively high abundance of tsRNAs has been noted in many high-throughput sequencing data sets, leading to largely correlative assumptions about their potential as biologically active entities. tRNAs are also the most modified RNAs in any cell type. Mutations in tRNA biogenesis factors including tRNA modification enzymes correlate with a variety of human disease syndromes. However, whether it is the lack of tRNAs or the activity of functionally relevant tsRNAs that are causative for human disease development remains to be elucidated. Here, we review the current knowledge in regard to tsRNAs biogenesis, including the impact of RNA modifications on tRNA stability and discuss the existing experimental evidence in support for the seemingly large functional spectrum being proposed for tsRNAs. We also argue that improved methodology allowing exact quantification and specific manipulation of tsRNAs will be necessary before developing these small RNAs into diagnostic biomarkers and when aiming to harness them for therapeutic purposes.

Transfer RNA Modification Enzymes from Thermophiles and Their Modified Nucleosides in tRNA.

To date, numerous modified nucleosides in tRNA as well as tRNA modification enzymes have been identified not only in thermophiles but also in mesophiles. Because most modified nucleosides in tRNA from thermophiles are common to those in tRNA from mesophiles, they are considered to work essentially in steps of protein synthesis at high temperatures. At high temperatures, the structure of unmodified tRNA will be disrupted. Therefore, thermophiles must possess strategies to stabilize tRNA structures. To this end, several thermophile-specific modified nucleosides in tRNA have been identified. Other factors such as RNA-binding proteins and polyamines contribute to the stability of tRNA at high temperatures. Thermus thermophilus, which is an extreme-thermophilic eubacterium, can adapt its protein synthesis system in response to temperature changes via the network of modified nucleosides in tRNA and tRNA modification enzymes. Notably, tRNA modification enzymes from thermophiles are very stable. Therefore, they have been utilized for biochemical and structural studies. In the future, thermostable tRNA modification enzymes may be useful as biotechnology tools and may be utilized for medical science.

The Emerging Field of Epitranscriptomics in Neurodevelopmental and Neuronal Disorders.

Analogous to DNA methylation and histone modifications, RNA modifications represent a novel layer of regulation of gene expression. The dynamic nature and increasing number of RNA modifications offer new possibilities to rapidly alter gene expression upon specific environmental changes. Recent lines of evidence indicate that modified RNA molecules and associated complexes regulating and “reading” RNA modifications play key roles in the nervous system of several organisms, controlling both, its development and function. Mutations in several human genes that modify transfer RNA (tRNA) have been linked to neurological disorders, in particular to intellectual disability. Loss of RNA modifications alters the stability of tRNA, resulting in reduced translation efficiency and generation of tRNA fragments, which can interfere with neuronal functions. Modifications present on messenger RNAs (mRNAs) also play important roles during brain development. They contribute to neuronal growth and regeneration as well as to the local regulation of synaptic functions. Hence, potential combinatorial effects of RNA modifications on different classes of RNA may represent a novel code to dynamically fine tune gene expression during brain function. Here we discuss the recent findings demonstrating the impact of modified RNAs on neuronal processes and disorders.

Mutations in Cytosine-5 tRNA Methyltransferases Impact Mobile Element Expression and Genome Stability at Specific DNA Repeats

The maintenance of eukaryotic genome stability is ensured by the interplay of transcriptional as well as post-transcriptional mechanisms that control recombination of repeat regions and the expression and mobility of transposable elements. We report here that mutations in two (cytosine-5) RNA methyltransferases, Dnmt2 and NSun2, impact the accumulation of mobile element-derived sequences and DNA repeat integrity in Drosophila. Loss of Dnmt2 function caused moderate effects under standard conditions, while heat shock exacerbated these effects. In contrast, NSun2 function affected mobile element expression and genome integrity in a heat shock-independent fashion. Reduced tRNA stability in both RCMT mutants indicated that tRNA-dependent processes affected mobile element expressionand DNA repeat stability. Importantly, further experiments indicated that complex formation with RNA could also contribute to theimpact of RCMT function on gene expression control. These results thus uncover a link between tRNA modification enzymes, the expression of repeat DNA, and genomic integrity.

Activation mode of the eukaryotic m2G10 tRNA methyltransferase Trm11 by its partner protein Trm112.

Post-transcriptional and post-translational modifications of factors involved in translation are very important for the control and accuracy of protein biosynthesis. Among these factors, tRNAs harbor the largest variety of grafted chemical structures, which participate in tRNA stability or mRNA decoding. Here, we focused on Trm112 protein, which associates with four different eukaryotic methyltransferases modifying tRNAs (Trm9 and Trm11) but also 18S-rRNA (Bud23) and translation termination factor eRF1 (Mtq2). In particular, we have investigated the role of Trm112 in the Trm11–Trm112 complex, which forms 2-methylguanosine at position 10 on several tRNAs and thereby is assumed to stabilize tRNA structure. We show that Trm112 is important for Trm11 enzymatic activity by influencing S-adenosyl-L-methionine binding and by contributing to tRNA binding. Using hydrogen-deuterium eXchange coupled to mass spectrometry, we obtained experimental evidences that the Trm11–Trm112 interaction relies on the same molecular bases as those described for other Trm112–methyltransferases complexes. Hence, all Trm112-dependent methyltransferases compete to interact with this partner.

A genetically encoded fluorescent tRNA is active in live-cell protein synthesis.

Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering.

Correlation between the stability of tRNA tertiary structure and the catalytic efficiency of a tRNA-modifying enzyme, archaeal tRNA-guanine transglycosylase.

In many archaeal tRNAs, archaeosine is found at position 15. During archaeosine biosynthesis, archaeal tRNA-guanine transglycosylase (ArcTGT) first replaces the guanine base at position 15 with 7-cyano-7-deazaguanine (preQ0). In this study, we investigated whether modified nucleosides in tRNA substrates would affect ArcTGT incorporation of preQ0. We prepared a series of hypomodified tRNAs(Ser)(GGA) from Escherichia coli strains lacking each tRNA-modifying enzyme. Measurement of ArcTGT kinetic parameters with the various tRNAs(Ser)(GGA) as substrates showed that the Km decreased due to the lack of modified nucleosides. The tRNAs(Ser)(GGA) melting profiles resulted in experimental evidence showing that each modified nucleoside in tRNA(Ser)(GGA) enhanced tRNA stability. Furthermore, the ArcTGT K(m) strongly correlated with the melting temperature (T(m)), suggesting that the unstable tRNA containing fewer modified nucleosides served as a better ArcTGT substrate. These results show that preQ0 incorporation into tRNA by ArcTGT takes place early in the archaeal tRNA modification process.

Crystal Structure of the Human tRNA m1A58 Methyltransferase–tRNA3Lys Complex: Refolding of Substrate tRNA Allows Access to the Methylation Target

Human tRNA3Lys is the primer for reverse transcription of HIV; the 3′ end is complementary to the primer-binding site on HIV RNA. The complementarity ends at the 18th base, A58, which in tRNA3Lys is modified to remove Watson–Crick pairing. Motivated to test the role of the modification in terminating the primer-binding sequence and thus limiting run-on transcription, we asked how the modification of RNA could be accomplished. tRNA m1A58 methyltransferase (m1A58 MTase) methylates N1 of A58, which is buried in the TΨC-loop of tRNA, from cofactor S-adenosyl-l-methionine. This conserved tRNA modification is essential for stability of initiator tRNA in Saccharomyces cerevisiae. Reported here, three structures of human tRNA m1A58 MTase in complex with human tRNA3Lys and the product S-adenosyl-l-homocysteine show a dimer of heterodimers in which each heterodimer comprises a catalytic chain, Trm61, and a homologous but noncatalytic chain, Trm6, repurposed as a tRNA-binding subunit that acts in trans; tRNAs bind across the dimer interface such that Trm6 from the opposing heterodimer brings A58 into the active site of Trm61. T-loop and D-loop are splayed apart showing how A58, normally buried in tRNA, becomes accessible for modification. This result has broad impact on our understanding of the mechanisms of modifying internal sites in folded tRNA. The structures serve as templates for design of inhibitors that could be used to test tRNA m1A58 MTase's impact on retroviral priming and transcription.