tRNA Function Research Articles

RNA versatility governs tRNA function: Why tRNA flexibility is essential beyond the translation cycle.

tRNAs undergo multiple conformational changes during the translation cycle that are required for tRNA translocation and proper communication between the ribosome and translation factors. Recent structural data on how destabilized tRNAs utilize the CCA-adding enzyme to proofread themselves put a spotlight on tRNA flexibility beyond the translation cycle. In analogy to tRNA surveillance, this review finds that other processes also exploit versatile tRNA folding to achieve, amongst others, specific aminoacylation, translational regulation by riboswitches or a block of bacterial translation. tRNA flexibility is thereby not restricted to the hinges utilized during translation. In contrast, the flexibility of tRNA is distributed all over its L-shape and is actively exploited by the tRNA-interacting partners to discriminate one tRNA from another. Since the majority of tRNA modifications also modulate tRNA flexibility it seems that cells devote enormous resources to tightly sense and regulate tRNA structure. This is likely required for error-free protein synthesis.

Role of codon usage and tRNA changes in rat cytomegalovirus latency and (re)activation.

Herpesviruses can remain in their hosts by establishing a latent infection with a low pattern of viral gene expression. Passage from latency to reactivation may occur under particular conditions such as immunosuppressive treatments or during fetal development, and often is accompanied by heavy pathologic sequelae. To investigate the molecular basis underlying herpesvirus latency and (re)activation, codon usage of rat cytomegalovirus was comparatively analyzed with respect to the rat codon usage. Two major points stand out as follows: (i) six codons - GCG (Ala), CCG (Pro), CGG (Arg), CGC (Arg), TCG (Ser), and ACG (Thr) - are rare in rat genes and intensively used in rat cytomegalovirus coding sequences; (ii) in many instances, the codons seldom used by the host are clustered along viral sequences coding for single amino acid repeats such as poly-Ala and poly-Thr stretches. The results indicate that rare host codons and their iteration along viral sequences might represent major constraints that lock rat cytomegalovirus translation in its host during the viral latent phase. Consequently, the data also suggest a link between rat cytomegalovirus quiescence/activation and the functional tRNA coadaptation phenomenon. Indeed, increases in minor tRNA species corresponding to rare rat codons mark rat cell proliferation and might rescue difficult viral translational contexts. Ala isoaccepting-tRNA (CGC) is reported as an example. On the whole, the present findings may contribute to explain how the molecular mechanisms that normally control host gene expression can silence/(re)activate viral gene expression, and might address research toward new approaches in anti-viral therapeutics.

Understanding the Effect of Post Transcriptional Modifications in the Anticodon Stem Loop of E.coli tRNA Arginine

Post-transcriptional modifications in the anticodon stem loop (ASL) of tRNAs are known to affect their conformation, thermal stability, ribosomal binding and decoding specificity. Escherichia coli uses only two tRNAArg isoacceptors to decode three of the six arginine codons (CGU, CGC and CGA). The ASLs of both the isoacceptors (tRNAArg1,2) contain naturally occurring modifications at position 34 (Inosine, I34) and 37 (2-methyladenosine, m2A37). In addition, tRNA Arg1 is post-transcriptionally modified to contain a 2-thiocytidine (s2C32) modification. To investigate the functional roles of these modifications, six ASL constructs, differing in their array of modifications, were analyzed using binding studies, as well as structural and computational methods. Ribosome filter binding assays showed that while I34, as expected, facilitates wobble codon binding, both s2C32 and m2A37 modulates that effect by negating binding to the rare CGA codon. Similar results were observed when a non-naturally occurring s2C32 was introduced for C32 in an unrelated S. cerevisiae tRNA ASLIle construct also containing I34 capable of wobble pairing. However, the solution structures show minor variation in the anticodon stem loop. The mechanism by which the modification affects codon binding was further investigated by molecular dynamics simulations. Using free-energy perturbation calculations we found that the s2C32 modification destabilizes the anticodon stem loop in solution in agreement with the experiments. Our simulations show that there is a free energy penalty for introducing s2C32 in ribosome bound tRNA explaining the reduction in binding observed experimentally. The simulation results are consistent with the experiments and provide a platform for gaining insights into the molecular explanation for how post-transcriptional modifications modulate tRNA function.

Control of Saccharomyces cerevisiae pre-tRNA processing by environmental conditions.

tRNA is essential for translation and decoding of the proteome. The yeast proteome responds to stress and tRNA biosynthesis contributes in this response by repression of tRNA transcription and alterations of tRNA modification. Here we report that the stress response also involves processing of pre-tRNA 3′ termini. By a combination of Northern analyses and RNA sequencing, we show that upon shift to elevated temperatures and/or to glycerol-containing medium, aberrant pre-tRNAs accumulate in yeast cells. For pre-tRNAUAUIle and pre-tRNAUUULys these aberrant forms are unprocessed at the 5′ ends, but they possess extended 3′ termini. Sequencing analyses showed that partial 3′ processing precedes 5′ processing for pre-tRNAUAUIle. An aberrant pre-tRNATyr that accumulates also possesses extended 3′ termini, but it is processed at the 5′ terminus. Similar forms of these aberrant pre-tRNAs are detected in the rex1Δ strain that is defective in 3′ exonucleolytic trimming of pre-tRNAs but are absent in the lhp1Δ mutant lacking 3′ end protection. We further show direct correlation between the inhibition of 3′ end processing rate and the stringency of growth conditions. Moreover, under stress conditions Rex1 nuclease seems to be limiting for 3′ end processing, by decreased availability linked to increased protection by Lhp1. Thus, our data document complex 3′ processing that is inhibited by stress in a tRNA-type and condition-specific manner. This stress-responsive tRNA 3′ end maturation process presumably contributes to fine-tune the levels of functional tRNA in budding yeast in response to environmental conditions.

Improving tRNAscan-SE Annotation Results via Ensemble Classifiers.

tRNAScan-SE is a tRNA detection program that is widely used for tRNA annotation; however, the false positive rate of tRNAScan-SE is unacceptable for large sequences. Here, we used a machine learning method to try to improve the tRNAScan-SE results. A new predictor, tRNA-Predict, was designed. We obtained real and pseudo-tRNA sequences as training data sets using tRNAScan-SE and constructed three different tRNA feature sets. We then set up an ensemble classifier, LibMutil, to predict tRNAs from the training data. The positive data set of 623 tRNA sequences was obtained from tRNAdb 2009 and the negative data set was the false positive tRNAs predicted by tRNAscan-SE. Our in silico experiments revealed a prediction accuracy rate of 95.1 % for tRNA-Predict using 10-fold cross-validation. tRNA-Predict was developed to distinguish functional tRNAs from pseudo-tRNAs rather than to predict tRNAs from a genome-wide scan. However, tRNA-Predict can work with the output of tRNAscan-SE, which is a genome-wide scanning method, to improve the tRNAscan-SE annotation results. The tRNA-Predict web server is accessible at http://datamining.xmu.edu.cn/∼gjs/tRNA-Predict.

Integrated tRNA, transcript, and protein profiles in response to steroid hormone signaling.

The accurate and efficient transfer of genetic information into amino acid sequences is carried out through codon–anticodon interactions between mRNA and tRNA, respectively. In this way, tRNAs function at the interface between gene expression and protein synthesis. Whether tRNA levels are dynamically regulated and to what degree tRNA abundance influences the cellular proteome remains largely unexplored. Here we profile tRNA, transcript and protein levels in Drosophila Kc167 cells, a plasmatocyte cell line that, upon treatment with 20-hydroxyecdysone, differentiates into macrophages. We find that high abundance tRNAs associate with codons that are overrepresented in the Kc167 cell proteome, whereas tRNAs that are in low supply associate with codons that are underrepresented. Ecdysone-induced differentiation of Kc167 cells leads to changes in mRNA codon usage in a manner consistent with the developmental progression of the cell. At both early and late time points, ecdysone treatment concomitantly increases the abundance of tRNAThr(CGU), which decodes a differentiation-associated codon that becomes enriched in the macrophage proteome. These results together suggest that tRNA levels may provide a meaningful regulatory mechanism for defining the cellular proteomic landscape.

Towards a comprehensive picture of alloacceptor tRNA remolding in metazoan mitochondrial genomes.

Remolding of tRNAs is a well-documented process in mitochondrial genomes that changes the identity of a tRNA. It involves a duplication of a tRNA gene, a mutation that changes the anticodon and the loss of the ancestral tRNA gene. The net effect is a functional tRNA that is more closely related to tRNAs of a different alloacceptor family than to tRNAs with the same anticodon in related species. Beyond being of interest for understanding mitochondrial tRNA function and evolution, tRNA remolding events can lead to artifacts in the annotation of mitogenomes and thus in studies of mitogenomic evolution. Therefore, it is important to identify and catalog these events. Here we describe novel methods to detect tRNA remolding in large-scale data sets and apply them to survey tRNA remolding throughout animal evolution. We identify several novel remolding events in addition to the ones previously mentioned in the literature. A detailed analysis of these remoldings showed that many of them are derived from ancestral events.

Expression of CRISPR/Cas single guide RNAs using small tRNA promoters.

The in vivo application of CRISPR/Cas-based DNA editing technology will require the development of efficient delivery methods that likely will be dependent on adeno-associated virus (AAV)-based viral vectors. However, AAV vectors have only a modest, ∼4.7-kb packaging capacity, which will necessitate the identification and characterization of highly active Cas9 proteins that are substantially smaller than the prototypic Streptococcus pyogenes Cas9 protein, which covers ∼4.2 kb of coding sequence, as well as the development of single guide RNA (sgRNA) expression cassettes substantially smaller than the current ∼360 bp size. Here, we report that small, ∼70-bp tRNA promoters can be used to express high levels of tRNA:sgRNA fusion transcripts that are efficiently and precisely cleaved by endogenous tRNase Z to release fully functional sgRNAs. Importantly, cells stably expressing functional tRNA:sgRNA precursors did not show a detectable change in the level of endogenous tRNA expression. This novel sgRNA expression strategy should greatly facilitate the construction of effective AAV-based Cas9/sgRNA vectors for future in vivo use.

RNA evolution conjectured from tRNA and riboswitches

The structures of tRNAs and riboswitches comprise distinct domains. A tRNA molecule is L-shaped, and each arm may reflect its evolution. The amino acid-nonspecific minihelix domain may have emerged before the amino acid- specific anticodon-containing domain was acquired. The glycine riboswitch of Bacillus subtilis functions in a glycine- independent manner in the presence of polyethylene glycol or ethylene glycol. The effect is dependent only on the existence of a terminator stem within the expression platform of the riboswitch and is indepen- dent of the aptamer domain. Similar to the hypothesis that explains the evolution of tRNA function, the specificity of riboswitch functional domains may have increased during evolution. Collectively, these views provide new perspectives on the evolution of RNA.

Controlling translation via modulation of tRNA levels.

Transfer RNAs (tRNAs) are critical adaptor molecules that carry amino acids to a messenger RNA (mRNA) template during protein synthesis. Although tRNAs have commonly been viewed as abundant 'house-keeping' RNAs, it is becoming increasingly clear that tRNA expression is tightly regulated. Depending on a cell's proliferative status, the pool of active tRNAs is rapidly changed, enabling distinct translational programs to be expressed in differentiated versus proliferating cells. Here, I highlight several post-transcriptional regulatory mechanisms that allow the expression or functions of tRNAs to be altered. Modulating the modification status or structural stability of individual tRNAs can cause those specific tRNA transcripts to selectively accumulate or be degraded. Decay generally occurs via the rapid tRNA decay pathway or by the nuclear RNA surveillance machinery. In addition, the CCA-adding enzyme plays a critical role in determining the fate of a tRNA. The post-transcriptional addition of CCA to the 3' ends of stable tRNAs generates the amino acid attachment site, whereas addition of CCACCA to unstable tRNAs prevents aminoacylation and marks the tRNA for degradation. In response to various stresses, tRNAs can accumulate in the nucleus or be further cleaved into small RNAs, some of which inhibit translation. By implementing these various post-transcriptional control mechanisms, cells are able to fine-tune tRNA levels to regulate subsets of mRNAs as well as overall translation rates.

Contribution of tRNA Met core flexibility to aminoacylation

The fidelity of protein synthesis is largely dominated by the correct recognition of transfer RNAs (tRNAs) by their cognate aminoacyl-tRNA synthetases (AARSs). Aminoacylation of each tRNA with its cognate amino acid is necessary to maintain the fidelity of the genetic code. Methionyl-tRNA synthetase (MetRS) aminoacylates both initator and elongator tRNAMet species for protein synthesis; the methionine-specifying CAU anticodon is a key contributor to tRNAMet identify. The communication between tRNAMet and MetRS is not well understood. We hypothesize that the core of tRNAMet is involved in dynamic communication between the tRNA anticodon and acceptor ends, and that such communication contributes to the rearrangement of the tRNA 3′-end to fit into the enzyme active site. Using both experimental and computational approaches, the effects of single nucleotide substitutions in the tRNAMet core and the impact they have on tRNA structure and function will be investigated.

Transfer RNA comes of age.

Transfer RNA comes of age.

Polyadenylation in E. coli: a 20 year odyssey.

Polyadenylation in E. coli: a 20 year odyssey.

Stabilization of eukaryotic ribosomal termination complexes by deacylated tRNA.

Stabilization of the ribosomal complexes plays an important role in translational control. Mechanisms of ribosome stabilization have been studied in detail for initiation and elongation of eukaryotic translation, but almost nothing is known about stabilization of eukaryotic termination ribosomal complexes. Here, we present one of the mechanisms of fine-tuning of the translation termination process in eukaryotes. We show that certain deacylated tRNAs, remaining in the E site of the ribosome at the end of the elongation cycle, increase the stability of the termination and posttermination complexes. Moreover, only the part of eRF1 recognizing the stop codon is stabilized in the A site of the ribosome, and the stabilization is not dependent on the hydrolysis of peptidyl-tRNA. The determinants, defining this property of the tRNA, reside in the acceptor stem. It was demonstrated by site-directed mutagenesis of tRNAVal and construction of a mini-helix structure identical to the acceptor stem of tRNA. The mechanism of this stabilization is different from the fixation of the unrotated state of the ribosome by CCA end of tRNA or by cycloheximide in the E site. Our data allow to reveal the possible functions of the isodecoder tRNAs in eukaryotes.

Cutting, dicing, healing and sealing: the molecular surgery of tRNA.

All organisms encode transfer RNAs (tRNAs) that are synthesized as precursor molecules bearing extra sequences at their 5' and 3' ends; some tRNAs also contain introns, which are removed by splicing. Despite commonality in what the ultimate goal is (i.e., producing a mature tRNA), mechanistically, tRNA splicing differs between Bacteria and Archaea or Eukarya. The number and position of tRNA introns varies between organisms and even between different tRNAs within the same organism, suggesting a degree of plasticity in both the evolution and persistence of modern tRNA splicing systems. Here we will review recent findings that not only highlight nuances in splicing pathways but also provide potential reasons for the maintenance of introns in tRNA. Recently, connections between defects in the components of the tRNA splicing machinery and medically relevant phenotypes in humans have been reported. These differences will be discussed in terms of the importance of splicing for tRNA function and in a broader context on how tRNA splicing defects can often have unpredictable consequences.

Modifying Mitochondrial tRNAs: Delivering What the Cell Needs

Protein synthesis is critically dependent on transfer (t)-RNAs, but the factors regulating tRNA function are poorly understood. In this issue of Cell Metabolism, Wei et al. (2015) show that Cdk5 regulatory subunit-associated protein-like-1 synchronizes mitochondrial and cytosolic translation in response to external stress, providing key insight into the pathogenesis of a common inherited mitochondrial disease.

TRNAs as antibiotic targets.

Transfer RNAs (tRNAs) are central players in the protein translation machinery and as such are prominent targets for a large number of natural and synthetic antibiotics. This review focuses on the role of tRNAs in bacterial antibiosis. We will discuss examples of antibiotics that target multiple stages in tRNA biology from tRNA biogenesis and modification, mature tRNAs, aminoacylation of tRNA as well as prevention of proper tRNA function by small molecules binding to the ribosome. Finally, the role of deacylated tRNAs in the bacterial “stringent response” mechanism that can lead to bacteria displaying antibiotic persistence phenotypes will be discussed.

Emerging roles of tRNA in adaptive translation, signalling dynamics and disease.

tRNAs, nexus molecules between mRNAs and proteins, have a central role in translation. Recent discoveries have revealed unprecedented complexity of tRNA biosynthesis, modification patterns, regulation and function. In this Review, we present emerging concepts regarding how tRNA abundance is dynamically regulated and how tRNAs (and their nucleolytic fragments) are centrally involved in stress signalling and adaptive translation, operating across a wide range of timescales. Mutations in tRNAs or in genes affecting tRNA biogenesis are also linked to complex human diseases with surprising heterogeneity in tissue vulnerability, and we highlight cell-specific aspects that modulate the disease penetrance of tRNA-based pathologies.

From end to end: tRNA editing at 5'- and 3'-terminal positions.

During maturation, tRNA molecules undergo a series of individual processing steps, ranging from exo- and endonucleolytic trimming reactions at their 5'- and 3'-ends, specific base modifications and intron removal to the addition of the conserved 3'-terminal CCA sequence. Especially in mitochondria, this plethora of processing steps is completed by various editing events, where base identities at internal positions are changed and/or nucleotides at 5'- and 3'-ends are replaced or incorporated. In this review, we will focus predominantly on the latter reactions, where a growing number of cases indicate that these editing events represent a rather frequent and widespread phenomenon. While the mechanistic basis for 5'- and 3'-end editing differs dramatically, both reactions represent an absolute requirement for generating a functional tRNA. Current in vivo and in vitro model systems support a scenario in which these highly specific maturation reactions might have evolved out of ancient promiscuous RNA polymerization or quality control systems.

Distribution and frequencies of post-transcriptional modifications in tRNAs

Functional tRNA molecules always contain a wide variety of post-transcriptionally modified nucleosides. These modifications stabilize tRNA structure, allow for proper interaction with other macromolecules and fine-tune the decoding of mRNAs during translation. Their presence in functionally important regions of tRNA is conserved in all domains of life. However, the identities of many of these modified residues depend much on the phylogeny of organisms the tRNAs are found in, attesting for domain-specific strategies of tRNA maturation. In this work we present a new tool, tRNAmodviz web server (http://genesilico.pl/trnamodviz) for easy comparative analysis and visualization of modification patterns in individual tRNAs, as well as in groups of selected tRNA sequences. We also present results of comparative analysis of tRNA sequences derived from 7 phylogenetically distinct groups of organisms: Gram-negative bacteria, Gram-positive bacteria, cytosol of eukaryotic single cell organisms, Fungi and Metazoa, cytosol of Viridiplantae, mitochondria, plastids and Euryarchaeota. These data update the study conducted 20 y ago with the tRNA sequences available at that time.