End Of tRNA Research Articles

Differential processing of small RNAs during endoplasmic reticulum stress

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) lumen due to the disruption of the homeostatic system of the ER leads to the induction of the ER stress response. Cellular stress-induced pathways globally transform genes expression on both the transcriptional and post-transcriptional levels with small RNA involvement as regulators of the stress response. The modulation of small RNA processing might represent an additional layer of a complex stress response program. However, it is poorly understood. Here, we studied changes in expression and small RNAs processing upon ER stress in Jurkat T-cells. Induced by ER-stress, depletion of miRNAs among small RNA composition was accompanied by a global decrease of 3′ mono-adenylated, mono-cytodinylated and a global increase of 3′ mono-uridinylated miRNA isoforms. We observed the specific subset of differentially expressed microRNAs, and also the dramatic induction of 32-nt tRNA fragments precisely phased to 5′ and 3′ ends of tRNA from a subset of tRNA isotypes. The induction of these tRNA fragments was linked to Angiogenin RNase, which mediates translation inhibition. Overall, the global perturbations of the expression and processing of miRNAs and tiRNAs were the most prominent features of small RNA transcriptome changes upon ER stress.

Defects in RNA metabolism in mitochondrial disease

The expression of mitochondrially-encoded genes requires the efficient processing of long precursor RNAs at the 5′ and 3′ ends of tRNAs, a process which, when disrupted, results in disease. Two such mutations reside within mt-tRNALeu(UUR); a m.3243A>G transition, which is the most common cause of MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), and m.3302A>G which often causes mitochondrial myopathy (MM). We used parallel analysis of RNA ends (PARE) that captures the 5′ terminal end of 5′-monophosphorylated mitochondrial RNAs to compare the effects of the m.3243A>G and m.3302A>G mutations on mitochondrial tRNA processing and downstream RNA metabolism. We confirmed previously identified RNA processing defects, identified common internal cleavage sites and new sites unique to the m.3243A>G mutants that do not correspond to transcript ends. These sites occur in regions of predicted RNA secondary structure, or are in close proximity to such regions, and may identify regions of importance to the processing of mtRNAs.

Dynamics of tRNA fragments and their targets in aging mammalian brain

Background: The progress of next-generation sequencing technologies has unveiled various non-coding RNAs that have previously been considered products of random degradation and attracted only minimal interest. Among small RNA families, microRNA (miRNAs) have traditionally been considered key post-transcriptional regulators. However, recent studies have reported evidence for widespread presence of fragments of tRNA molecules (tRFs) across a range of organisms and tissues, and of tRF involvement in Argonaute complexes. Methods:To elucidate potential tRF functionality, we compared available RNA sequencing datasets derived from the brains of young, mid-aged and old rats. Using sliding 7-mer windows along a tRF, we searched for putative seed sequences with high numbers of conserved complementary sites within 3' UTRs of 23 vertebrate genomes. We analyzed Gene Ontology term enrichment of predicted tRF targets and compared their transcript levels with targets of miRNAs in the context of age. Results and Discussion: We detected tRFs originating from 3’- and 5’-ends of tRNAs in rat brains at significant levels. These fragments showed dynamic changes: 3’ tRFs monotonously increased with age, while 5’ tRFs displayed less consistent patterns. Furthermore, 3’ tRFs showed a narrow size range compared to 5’ tRFs, suggesting a difference in their biogenesis mechanisms. Similar to our earlier results in Drosophila and compatible with other experimental findings, we found “seed” sequence locations on both ends of different tRFs. Putative targets of these fragments were found to be enriched in neuronal and developmental functions. Comparison of tRFs and miRNAs increasing in abundance with age revealed small, but distinct changes in brain target transcript levels for these two types of small RNA, with the higher proportion of tRF targets decreasing with age. We also illustrated the utility of tRF analysis for annotating tRNA genes in sequenced genomes.

Initiator tRNA genes template the 3' CCA end at high frequencies in bacteria.

BackgroundWhile the CCA sequence at the mature 3′ end of tRNAs is conserved and critical for translational function, a genetic template for this sequence is not always contained in tRNA genes. In eukaryotes and Archaea, the CCA ends of tRNAs are synthesized post-transcriptionally by CCA-adding enzymes. In Bacteria, tRNA genes template CCA sporadically.ResultsIn order to understand the variation in how prokaryotic tRNA genes template CCA, we re-annotated tRNA genes in tRNAdb-CE database version 0.8. Among 132,129 prokaryotic tRNA genes, initiator tRNA genes template CCA at the highest average frequency (74.1%) over all functional classes except selenocysteine and pyrrolysine tRNA genes (88.1% and 100% respectively). Across bacterial phyla and a wide range of genome sizes, many lineages exist in which predominantly initiator tRNA genes template CCA. Convergent and parallel retention of CCA templating in initiator tRNA genes evolved in independent histories of reductive genome evolution in Bacteria. Also, in a majority of cyanobacterial and actinobacterial genera, predominantly initiator tRNA genes template CCA. We also found that a surprising fraction of archaeal tRNA genes template CCA.ConclusionsWe suggest that cotranscriptional synthesis of initiator tRNA CCA 3′ ends can complement inefficient processing of initiator tRNA precursors, “bootstrap” rapid initiation of protein synthesis from a non-growing state, or contribute to an increase in cellular growth rates by reducing overheads of mass and energy to maintain nonfunctional tRNA precursor pools. More generally, CCA templating in structurally non-conforming tRNA genes can afford cells robustness and greater plasticity to respond rapidly to environmental changes and stimuli.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3314-x) contains supplementary material, which is available to authorized users.

Dynamics of tRNA fragments and their targets in aging mammalian brain.

Background:The progress of next-generation sequencing technologies has unveiled various non-coding RNAs that have previously been considered products of random degradation and attracted only minimal interest. Among small RNA families, microRNA (miRNAs) have traditionally been considered key post-transcriptional regulators. However, recent studies have reported evidence for widespread presence of fragments of tRNA molecules (tRFs) across a range of organisms and tissues, and of tRF involvement in Argonaute complexes. Methods:To elucidate potential tRF functionality, we compared available RNA sequencing datasets derived from the brains of young, mid-aged and old rats. Using sliding 7-mer windows along a tRF, we searched for putative seed sequences with high numbers of conserved complementary sites within 3' UTRs of 23 vertebrate genomes. We analyzed Gene Ontology term enrichment of predicted tRF targets and compared their transcript levels with targets of miRNAs in the context of age. Results and Discussion:We detected tRFs originating from 3'- and 5'-ends of tRNAs in rat brains at significant levels. These fragments showed dynamic changes: 3' tRFs monotonously increased with age, while 5' tRFs displayed less consistent patterns. Furthermore, 3' tRFs showed a narrow size range compared to 5' tRFs, suggesting a difference in their biogenesis mechanisms. Similar to our earlier results in Drosophila and compatible with other experimental findings, we found "seed" sequence locations on both ends of different tRFs. Putative targets of these fragments were found to be enriched in neuronal and developmental functions. Comparison of tRFs and miRNAs increasing in abundance with age revealed small, but distinct changes in brain target transcript levels for these two types of small RNA, with the higher proportion of tRF targets decreasing with age. We also illustrated the utility of tRF analysis for annotating tRNA genes in sequenced genomes.

Small RNAs in Sperm, Affected by Diet, Transmit Information to Offspring

A class of small RNAs derived from tRNAs has a distinct profile in mice fed unhealthy diets, suggests two new studies. These RNAs may be involved in the intergenerational inheritance of metabolic states. Studies in the last several years have suggested that fathers can pass down metabolic information to offspring. For instance, male mice fed either a low-protein or high-fat diet produce offspring with metabolic abnormalities. In flies and worms, piRNAs—typically 25 to 33 nucleotides in length— have been implicated in transgenerational inheritance. But whether small RNAs have a similar role in mammals has been unclear. Previous research found that a class of small RNAs is abundant in mouse sperm, tRNA-derived small RNAS (tsRNAS). tRNAs are derived mainly from the 5 ends of tRNAs and have been suggested to have regulatory roles in various cell types. The new findings suggest that tsRNA profiles are different in sperm from males subjected to either a low-protein or high-fat diet. In one study, Qi Zhou and colleagues [1] fed mice a high-fat diet and isolated small RNAs from their sperm including a 30to 40-nucleotide fraction that consisted predominantly of tsRNAs. Injection of this RNA fraction into normal zygotes resulted in altered expression of numerous genes, including those involved in metabolic pathways; this RNA fraction also affected the metabolic phenotype of F1 offspring. In a second study, Oliver Rando and colleagues [2] provide evidence that tsRNAs are delivered to sperm via epididymosomes: vesicles that fuse with sperm as they transit through the epididymis. A protein-restricted diet resulted in increased levels of glycine tsRNAs, which seem to have a regulatory role in the embryo—for instance, interfering with a glycine tsRNAaffected expression of a set of about 70 genes in embryonic stem cells. Curiously, these genes are highly expressed in the preimplantation embryos and are regulated by the same DNA element (the long terminal repeat of the murine endogenous retroelement, MERVL). Injection of a synthetic oligo of this tsRNA affected expression of a similar set of genes in the early embryo. Whether injection of this oligo affected the metabolic phenotype of the offspring was not tested. The findings suggest that diet can affect tsRNAs in sperm and that these tsRNAs may have a role in intergenerational inheritance. But other factors may also come into play; for instance, other molecules were also altered in the sperm from males fed unhealthy diets, such as the let-7 small regulatory RNA. Another recent study in humans has suggested the piRNA profiles and DNA methylation in sperm is affected by diet [3].

Native Chemical Ligation of Hydrolysis-Resistant 3'-NH-Cysteine-Modified RNA.

Hydrolysis-resistant RNA-peptide conjugates that contain a 3'-NH linkage between the adenosine ribose and the C-terminal carboxyl group of a peptide moiety instead of the natural ester mimic acylated tRNA termini. Their detailed preparation that combines solid-phase oligonucleotide synthesis and bioconjugation is described here. The key step is native chemical ligation (NCL) of 3'-NH-cysteine-modified RNA to highly soluble peptide thioesters. These hydrolysis-resistant 3'-NH-peptide-modified RNAs, containing the universally conserved 3'-CCA end of tRNA, are biologically active and can bind to the ribosome. They can be used as valuable probes for structural and functional studies of the ribosomal elongation cycle.

Angiogenin Interacts with Heat Shock Factor 1

Angiogenin (ANG) and heat shock factor 1 (HSF1) function in distinct pathways to mediate cellular stress. HSF1, an evolutionarily highly conserved protein, plays a role in a multitude of cellular processes that includes responses to cell stress as well as development, metabolism, and aging. Upon stress in cells, monomeric HSF1 trimerizes, localizes to the nucleus, binds to heat shock elements (HSE), and activates transcription of chaperones, co-chaperones, and ubiquitin. ANG, a vertebrate-specific ribonuclease with potent angiogenic activity, possesses both RNA- and DNA-binding capacity. During cell stress it is activated to cleave the anticodon loops and CCA-3' ends of tRNA in order to stall translation, however, the protein and DNA targets of ANG that facilitate changes in gene expression are not known. In independent studies, both ANG and HSF1 have been found to bind to DNA sequences within pericentromeres. The goal of this study was to determine if ANG and HSF1 interact directly. Using pull-down experiments, we demonstrated a specific interaction between ANG and two distinct forms of HSF1. The mechanistic relationship between ANG and HSF1 is yet undiscovered and is the focus of our current work.

Reflections on the 20th anniversary of RNA.

Reflections on the 20th anniversary of RNA.

My encounter with RNA.

My encounter with RNA.

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.

The Kinetics of Nascent Protein Folding upon Release from the Ribosome

Very little is known about the way proteins attain their native structure within the context of the living cell. In addition to the ribosome's well-established role in peptide bond formation, recent studies suggest that ribosomes play an important role in the early stages of protein folding in the cell and may be crucial for the production of folded bioactive proteins. Importantly, little is known about the impact of the mechanism of protein release from the ribosome on the attainment of a correctly folded conformation. Here, we present a kinetic study on the release time-course of fully synthesized ribosome-bound nascent proteins upon addition of the antibiotic puromycin. We focus these studies on the E. coli globin ApoHmpH. By time-resolved gel electrophoresis, we are able to follow puromycin's hydrolysis of the ester bond linking nascent polypeptides to the 3’ end of tRNA. Steady-state fluorescence anisotropy allows us to follow the escape and folding of ApoHmpH from the ribosome. Finally, time decay fluorescence anisotropy analysis in the frequency domain complements the above techniques by providing insights into the local motions experienced by the nascent protein before and after release from the ribosome. Under experimental conditions where puromycin reacts at rates comparable to the naturally occurring release factors, we show that protein release from the ribosome is rate-limited by the C-terminal ester bond cleavage, and that escape from the ribosome and completion of folding occur quickly following this step. This result shows that the ribosomal context promotes a particularly “temporally efficient” folding upon nascent protein release. An important consequence of this phenomenon is the prevention of undesirable diffusion- and concentration-dependent phenomena such as aggregation.

Diverse cell stresses induce unique patterns of tRNA up- and down-regulation: tRNA-seq for quantifying changes in tRNA copy number.

Emerging evidence points to roles for tRNA modifications and tRNA abundance in cellular stress responses. While isolated instances of stress-induced tRNA degradation have been reported, we sought to assess the effects of stress on tRNA levels at a systems level. To this end, we developed a next-generation sequencing method that exploits the paucity of ribonucleoside modifications at the 3′-end of tRNAs to quantify changes in all cellular tRNA molecules. Application of this tRNA-seq method to Saccharomyces cerevisiae identified all 76 expressed unique tRNA species out of 295 coded in the yeast genome, including all isoacceptor variants, with highly precise relative (fold-change) quantification of tRNAs. In studies of stress-induced changes in tRNA levels, we found that oxidation (H2O2) and alkylation (methylmethane sulfonate, MMS) stresses induced nearly identical patterns of up- and down-regulation for 58 tRNAs. However, 18 tRNAs showed opposing changes for the stresses, which parallels our observation of signature reprogramming of tRNA modifications caused by H2O2 and MMS. Further, stress-induced degradation was limited to only a small proportion of a few tRNA species. With tRNA-seq applicable to any organism, these results suggest that translational control of stress response involves a contribution from tRNA abundance.

An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center.

The Watson-Crick base pairs between the 3'-terminal end of tRNAs and ribosomal RNA in the peptidyl transferase center are universally conserved. Here, we report that the introduction of compensatory mutations to Escherichia coli RNAs in this site leads to an orthogonal system independent of the wild-type counterpart, as demonstrated via the production of two peptide sequences from a single mRNA. This work thus identifies a new way to reprogram the genetic code.

Fusion with Anticodon Binding Domain of GluRS is Not Sufficient to Alter the Substrate Specificity of a Chimeric Glu-Q-RS

Glutamyl-queuosine-tRNA(Asp) synthetase (Glu-Q-RS) is a paralog of glutamyl-tRNA synthetase (GluRS) and is found in more than forty species of proteobacteria, cyanobacteria, and actinobacteria. Glu-Q-RS shows striking structural similarity with N-terminal catalytic domain of GluRS (NGluRS) but it lacks the C-terminal anticodon binding domain (CGluRS). In spite of structural similarities, Glu-Q-RS and NGluRS differ in their functional properties. Glu-Q-RS glutamylates the Q34 nucleotide of the anticodon of tRNA(Asp) whereas NGluRS constitutes the catalytic domain of GluRS catalyzing the transfer of Glu on the acceptor end of tRNA(Glu). Since NGluRS is able to catalyze aminoacylation of only tRNA(Glu) the glutamylation capacity of tRNA(Asp) by Glu-Q-RS is surprising. To understand the substrate specificity of Glu-Q-RS we undertook a systemic approach by investigating the biophysical and biochemical properties of the NGluRS (1-301), CGluRS (314-471) and Glu-Q-RS-CGluRS, (1-298 of Glu-Q-RS fused to 314-471 from GluRS). Circular dichroism, fluorescence spectroscopy and differential scanning calorimetry analyses revealed absence of N-terminal domain (1-298 of Glu-Q-RS) and C-terminal domain (314-471 from GluRS) communication in chimera, in contrast to the native full length GluRS. The chimeric Glu-Q-RS is still able to aminoacylate tRNA(Asp) but has also the capacity to bind tRNA(Glu). However the chimeric protein is unable to aminoacylate tRNA(Glu) probably as a consequence of the lack of domain-domain communication.

Partitioning of the Nuclear and Mitochondrial tRNA 3′-End Processing Activities between Two different Proteins in Schizosaccharomyces pombe

tRNase Z is an essential endonuclease responsible for tRNA 3'-end maturation. tRNase Z exists in a short form (tRNase Z(S)) and a long form (tRNase Z(L)). Prokaryotes have only tRNase Z(S), whereas eukaryotes can have both forms of tRNase Z. Most eukaryotes characterized thus far, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and humans, contain only one tRNase Z(L) gene encoding both nuclear and mitochondrial forms of tRNase Z(L). In contrast, Schizosaccharomyces pombe contains two essential tRNase Z(L) genes (trz1 and trz2) encoding two tRNase Z(L) proteins, which are targeted to the nucleus and mitochondria, respectively. Trz1 protein levels are notably higher than Trz2 protein levels. Here, using temperature-sensitive mutants of trz1 and trz2, we provide in vivo evidence that trz1 and trz2 are involved in nuclear and mitochondrial tRNA 3'-end processing, respectively. In addition, trz2 is also involved in generation of the 5'-ends of other mitochondrial RNAs, whose 5'-ends coincide with the 3'-end of tRNA. Thus, our results provide a rare example showing partitioning of the nuclear and mitochondrial tRNase Z(L) activities between two different proteins in S. pombe. The evolution of two tRNase Z(L) genes and their differential expression in fission yeast may avoid toxic off-target effects.

Structural Studies of a Bacterial tRNAHIS Guanylyltransferase (Thg1)-Like Protein, with Nucleotide in the Activation and Nucleotidyl Transfer Sites

All nucleotide polymerases and transferases catalyze nucleotide addition in a 5′ to 3′ direction. In contrast, tRNAHis guanylyltransferase (Thg1) enzymes catalyze the unusual reverse addition (3′ to 5′) of nucleotides to polynucleotide substrates. In eukaryotes, Thg1 enzymes use the 3′–5′ addition activity to add G−1 to the 5′-end of tRNAHis, a modification required for efficient aminoacylation of the tRNA by the histidyl-tRNA synthetase. Thg1-like proteins (TLPs) are found in Archaea, Bacteria, and mitochondria and are biochemically distinct from their eukaryotic Thg1 counterparts TLPs catalyze 5′-end repair of truncated tRNAs and act on a broad range of tRNA substrates instead of exhibiting strict specificity for tRNAHis. Taken together, these data suggest that TLPs function in distinct biological pathways from the tRNAHis maturation pathway, perhaps in tRNA quality control. Here we present the first crystal structure of a TLP, from the gram-positive soil bacterium Bacillus thuringiensis (BtTLP). The enzyme is a tetramer like human THG1, with which it shares substantial structural similarity. Catalysis of the 3′–5′ reaction with 5′-monophosphorylated tRNA necessitates first an activation step, generating a 5′-adenylylated intermediate prior to a second nucleotidyl transfer step, in which a nucleotide is transferred to the tRNA 5′-end. Consistent with earlier characterization of human THG1, we observed distinct binding sites for the nucleotides involved in these two steps of activation and nucleotidyl transfer. A BtTLP complex with GTP reveals new interactions with the GTP nucleotide in the activation site that were not evident from the previously solved structure. Moreover, the BtTLP-ATP structure allows direct observation of ATP in the activation site for the first time. The BtTLP structural data, combined with kinetic analysis of selected variants, provide new insight into the role of key residues in the activation step.

Small RNAs derived from the 5′ end of tRNA can inhibit protein translation in human cells

Recently, it has been shown that tRNA molecules can be processed into small RNAs that are derived from both the 5′ and 3′ termini. To date, the function of these tRNA fragments (tRFs) derived from the 5′ end of tRNA has not been investigated in depth. We present evidence that conserved residues in tRNA, present in all 5′ tRFs, can inhibit the process of protein translation without the need for complementary target sites in the mRNA. These results implicate 5′ tRFs in a new mechanism of gene regulation by small RNAs in human cells.

Exploring the Kinetics of Protein Birth

Fidelity and efficiency in protein folding are essential to sustain cell life. Overall however, very little is known about the way proteins are able to attain their native structure within the context of the cell. In addition to the ribosome's well-established role in peptide bond formation, recent studies suggest that ribosomes may have strong influence on the early stages of protein folding in the cell and may be crucial for the production of folded unaggregated proteins. The conformational changes that occur within a nascent protein during its release from the ribosome have yet to be elucidated. Here, we present a kinetic study on the release time-course of ribosome bound model proteins upon addition of the antibiotic puromycin. By time-resolved gel electrophoresis, we are able to discern that puromycin's hydrolysis of the ester bond linking nascent polypeptides to the 3' end of tRNA occurs quickly. Steady-state fluorescence anisotropy reveals the presence of two additional slower kinetic phases. Finally, time decay fluorescence anisotropy analysis complements the above results by providing insights into the local motions experienced by the nascent protein during different stages of the protein birth process.

A Pseudo-tRNA Modulates Antibiotic Resistance in Bacillus cereus

Bacterial genomic islands are often flanked by tRNA genes, which act as sites for the integration of foreign DNA into the host chromosome. For example, Bacillus cereus ATCC14579 contains a pathogenicity island flanked by a predicted pseudo-tRNA, tRNAOther, which does not function in translation. Deletion of tRNAOther led to significant changes in cell wall morphology and antibiotic resistance and was accompanied by changes in the expression of numerous genes involved in oxidative stress responses, several of which contain significant complementarities to sequences surrounding tRNAOther. This suggested that tRNAOther might be expressed as part of a larger RNA, and RACE analysis subsequently confirmed the existence of several RNA species that significantly extend both the 3′ and 5′-ends of tRNAOther. tRNAOther expression levels were found to be responsive to changes in extracellular iron concentration, consistent with the presence of three putative ferric uptake regulator (Fur) binding sites in the 5′ leader region of one of these larger RNAs. Taken together with previous data, this study now suggests that tRNAOther may function by providing a tRNA-like structural element within a larger regulatory RNA. These findings illustrate that while integration of genomic islands often leaves tRNA genes intact and functional, in other instances inactivation may generate tRNA-like elements that are then recruited to other functions in the cell.