Dynamic queuosine changes in tRNA couple nutrient levels to codon choice in Trypanosoma brucei.

Every type of nucleic acid in cells undergoes programmed chemical post-transcriptional modification. Generally, modification enzymes use substrates derived from intracellular metabolism, one exception is queuine (q)/queuosine (Q), which eukaryotes obtain from their environment; made by bacteria and ultimately taken into eukaryotic cells via currently unknown transport systems. Here, we use a combination of molecular, cell biology and biophysical approaches to show that in Trypanosoma brucei tRNA Q levels change dynamically in response to concentration variations of a sub-set of amino acids in the growth media. Most significant were variations in tyrosine, which at low levels lead to increased Q content for all the natural tRNAs substrates of tRNA-guanine transglycosylase (TGT). Such increase results from longer nuclear dwell time aided by retrograde transport following cytoplasmic splicing. In turn high tyrosine levels lead to rapid decrease in Q content. Importantly, the dynamic changes in Q content of tRNAs have negligible effects on global translation or growth rate but, at least, in the case of tRNATyr it affected codon choice. These observations have implications for the occurrence of other tunable modifications important for 'normal' growth, while connecting the intracellular localization of modification enzymes, metabolites and tRNAs to codon selection and implicitly translational output.

Increased expression of tryptophan and tyrosine tRNAs elevates stop codon readthrough of reporter systems in human cell lines.

Regulation of translation via stop codon readthrough (SC-RT) expands not only tissue-specific but also viral proteomes in humans and, therefore, represents an important subject of study. Understanding this mechanism and all involved players is critical also from a point of view of prospective medical therapies of hereditary diseases caused by a premature termination codon. tRNAs were considered for a long time to be just passive players delivering amino acid residues according to the genetic code to ribosomes without any active regulatory roles. In contrast, our recent yeast work identified several endogenous tRNAs implicated in the regulation of SC-RT. Swiftly emerging studies of human tRNA-ome also advocate that tRNAs have unprecedented regulatory potential. Here, we developed a universal U6 promotor-based system expressing various human endogenous tRNA iso-decoders to study consequences of their increased dosage on SC-RT employing various reporter systems in vivo. This system combined with siRNA-mediated downregulations of selected aminoacyl-tRNA synthetases demonstrated that changing levels of human tryptophan and tyrosine tRNAs do modulate efficiency of SC-RT. Overall, our results suggest that tissue-to-tissue specific levels of selected near-cognate tRNAs may have a vital potential to fine-tune the final landscape of the human proteome, as well as that of its viral pathogens.

Comparative tRNA sequencing and RNA mass spectrometry for surveying tRNA modifications.

Chemical modifications of the nucleosides that comprise transfer RNAs are diverse. However, the structure, location and extent of modifications have been systematically charted in very few organisms. Here, we describe an approach in which rapid prediction of modified sites through reverse transcription-derived signatures in high-throughput transfer RNA-sequencing (tRNA-seq) data is coupled with identification of tRNA modifications through RNA mass spectrometry. Comparative tRNA-seq enabled prediction of several Vibrio cholerae modifications that are absent from Escherichia coli and also revealed the effects of various environmental conditions on V. cholerae tRNA modification. Through RNA mass spectrometric analyses, we showed that two of the V. cholerae-specific reverse transcription signatures reflected the presence of a new modification (acetylated acp3U (acacp3U)), while the other results from C-to-Ψ RNA editing, a process not described before. These findings demonstrate the utility of this approach for rapid surveillance of tRNA modification profiles and environmental control of tRNA modification.

Crystal structure of Nanoarchaeum equitans tyrosyl-tRNA synthetase and its aminoacylation activity toward tRNATyr with an extra guanosine residue at the 5'-terminus.

tRNATyr of Nanoarchaeum equitans has a remarkable feature with an extra guanosine residue at the 5'-terminus. However, the N. equitans tRNATyr mutant without extra guanosine at the 5'-end was tyrosylated by tyrosyl-tRNA synthase (TyrRS). We solved the crystal structure of N. equitans TyrRS at 2.80 Å resolution. By comparing the present solved structure with the complex structures TyrRS with tRNATyr of Thermus thermophilus and Methanocaldococcus jannaschii, an arginine substitution mutant of N. equitans TyrRS at Ile200 (I200R), which is the putative closest candidate to the 5'-phosphate of C1 of N. equitans tRNATyr, was prepared. The I200R mutant tyrosylated not only wild-type tRNATyr but also the tRNA without the G-1 residue. Further tyrosylation analysis revealed that the second base of the anticodon (U35), discriminator base (A73), and C1:G72 base pair are strong recognition sites.

Unusual noncanonical intron editing is important for tRNA splicing in Trypanosoma brucei.

In cells, tRNAs are synthesized as precursor molecules bearing extra sequences at their 5' and 3' ends. Some tRNAs also contain introns, which, in archaea and eukaryotes, are cleaved by an evolutionarily conserved endonuclease complex that generates fully functional mature tRNAs. In addition, tRNAs undergo numerous posttranscriptional nucleotide chemical modifications. In Trypanosoma brucei, the single intron-containing tRNA (tRNA(Tyr)GUA) is responsible for decoding all tyrosine codons; therefore, intron removal is essential for viability. Using molecular and biochemical approaches, we show the presence of several noncanonical editing events, within the intron of pre-tRNA(Tyr)GUA, involving guanosine-to-adenosine transitions (G to A) and an adenosine-to-uridine transversion (A to U). The RNA editing described here is required for proper processing of the intron, establishing the functional significance of noncanonical editing with implications for tRNA processing in the deeply divergent kinetoplastid lineage and eukaryotes in general.

Conditional accumulation of toxic tRNAs to cause amino acid misincorporation.

To develop a system for conditional amino acid misincorporation, we engineered tRNAs in the yeast Saccharomyces cerevisiae to be substrates of the rapid tRNA decay (RTD) pathway, such that they accumulate when RTD is turned off. We used this system to test the effects on growth of a library of tRNASer variants with all possible anticodons, and show that many are lethal when RTD is inhibited and the tRNA accumulates. Using mass spectrometry, we measured serine misincorporation in yeast containing each of six tRNA variants, and for five of them identified hundreds of peptides with serine substitutions at the targeted amino acid sites. Unexpectedly, we found that there is not a simple correlation between toxicity and the level of serine misincorporation; in particular, high levels of serine misincorporation can occur at cysteine residues without obvious growth defects. We also showed that toxic tRNAs can be used as a tool to identify sequence variants that reduce tRNA function. Finally, we generalized this method to another tRNA species, and generated conditionally toxic tRNATyr variants in a similar manner. This method should facilitate the study of tRNA biology and provide a tool to probe the effects of amino acid misincorporation on cellular physiology.

The complete mitochondrial genome of Sarcoptes scabiei var. nyctereutis from the Japanese raccoon dog: Prediction and detection of two transfer RNAs (tRNA-A and tRNA-Y).

Sarcoptes scabiei (Acari: Sarcoptidae) causes a common contagious skin disease that affects many mammals. Here, the complete mitochondrial genome of a mite, S. scabiei var. nyctereutis, from Japanese wild raccoon dogs was analyzed. The 13,837bp circular genome contained 13 protein-coding genes, two rRNA genes, and 22 tRNA genes. For the first time, two tRNAs (alanine and tyrosine), that were thought to be absent in scabies mites from other animals, were predicted to have short, non-cloverleaf structures by in silico annotation and detected by RT-PCR, sequencing, and northern analysis. The mitochondrial genome structure of S. scabiei is similar to that of Psoroptes cuniculi and Dermatophagoides farinae. While small and unusual tRNA genes seem to be common among acariform mites, further experimental evidence for their presence is needed. Furthermore, through an analysis of the cox1 gene, we have provided new evidence to confirm the transmission of this mite between different animal hosts.

CLP1 links tRNA metabolism to progressive motor-neuron loss.

CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1(K/K)) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1(K/K) mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53.

Retrograde nuclear transport from the cytoplasm is required for tRNATyr maturation in T. brucei.

Retrograde transport of tRNAs from the cytoplasm to the nucleus was first described in Saccharomyces cerevisiae and most recently in mammalian systems. Although the function of retrograde transport is not completely clear, it plays a role in the cellular response to changes in nutrient availability. Under low nutrient conditions tRNAs are sent from the cytoplasm to nucleus and presumably remain in storage there until nutrient levels improve. However, in S. cerevisiae tRNA retrograde transport is constitutive and occurs even when nutrient levels are adequate. Constitutive transport is important, at least, for the proper maturation of tRNAPhe, which undergoes cytoplasmic splicing, but requires the action of a nuclear modification enzyme that only acts on a spliced tRNA. A lingering question in retrograde tRNA transport is whether it is relegated to S. cerevisiae and multicellular eukaryotes or alternatively, is a pathway with deeper evolutionary roots. In the early branching eukaryote Trypanosoma brucei, tRNA splicing, like in yeast, occurs in the cytoplasm. In the present report, we have used a combination of cell fractionation and molecular approaches that show the presence of significant amounts of spliced tRNATyr in the nucleus of T. brucei. Notably, the modification enzyme tRNA-guanine transglycosylase (TGT) localizes to the nucleus and, as shown here, is not able to add queuosine (Q) to an intron-containing tRNA. We suggest that retrograde transport is partly the result of the differential intracellular localization of the splicing machinery (cytoplasmic) and a modification enzyme, TGT (nuclear). These findings expand the evolutionary distribution of retrograde transport mechanisms to include early diverging eukaryotes, while highlighting its importance for queuosine biosynthesis.

Isoacceptor specific characterization of tRNA aminoacylation and misacylation in vivo.

Amino acid misincorporation during protein synthesis occurs due to misacylation of tRNAs or defects in decoding at the ribosome. While misincorporation of amino acids has been observed in a variety of contexts, less work has been done to directly assess the extent to which specific tRNAs are misacylated in vivo, and the identity of the misacylated amino acid moiety. Here we describe tRNA isoacceptor specific aminoacylation profiling (ISAP), a method to identify and quantify the amino acids attached to a tRNA species in vivo. ISAP allows compilation of aminoacylation profiles for specific isoacceptors tRNAs. To demonstrate the efficacy and broad applicability of ISAP, tRNAPhe and tRNATyr species were isolated from total aminoacyl-tRNA extracted from both yeast and Escherichia coli. Isolated aminoacyl-tRNAs were washed until free of detectable unbound amino acid and subsequently deacylated. Free amino acids from the deacylated fraction were then identified and quantified by mass spectrometry. Using ISAP allowed quantification of the effects of quality control on the accumulation of misacylated tRNA species under different growth conditions.

Spectrophotometric assays for monitoring tRNA aminoacylation and aminoacyl-tRNA hydrolysis reactions.

Aminoacyl-tRNA synthetases play a central role in protein synthesis, catalyzing the attachment of amino acids to their cognate tRNAs. Here, we describe a spectrophotometric assay for tyrosyl-tRNA synthetase in which the Tyr-tRNA product is cleaved, regenerating the tRNA substrate. As tRNA is the limiting substrate in the assay, recycling it substantially increases the sensitivity of the assay while simultaneously reducing its cost. The tRNA aminoacylation reaction is monitored spectrophotometrically by coupling the production of AMP to the conversion of NAD+ to NADH. We have adapted the tyrosyl-tRNA synthetase assay to monitor: (1) aminoacylation of tRNA by l- or d-tyrosine, (2) cyclodipeptide formation by cyclodipeptide synthases, (3) hydrolysis of d-aminoacyl-tRNAs by d-tyrosyl-tRNA deacylase, and (4) post-transfer editing by aminoacyl-tRNA synthetases. All of these assays are continuous and homogenous, making them amenable for use in high-throughput screens of chemical libraries. In the case of the cyclodipeptide synthase, d-tyrosyl-tRNA deacylase, and post-transfer editing assays, the aminoacyl-tRNAs are generated in situ, avoiding the need to synthesize and purify aminoacyl-tRNA substrates prior to performing the assays. Lastly, we describe how the tyrosyl-tRNA synthetase assay can be adapted to monitor the activity of other aminoacyl-tRNA synthetases and how the approach to regenerating the tRNA substrate can be used to increase the sensitivity and decrease the cost of commercially available aminoacyl-tRNA synthetase assays.

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF.

Pseudouridine is found in almost all cellular ribonucleic acids (RNAs). Of the multiple characteristics attributed to pseudouridine, making messenger RNAs (mRNAs) highly translatable and non-immunogenic is one such feature that directly implicates this modification in protein synthesis. We report the existence of pseudouridine in the anticodon of Escherichia coli tyrosine transfer RNAs (tRNAs) at position 35. Pseudouridine was verified by multiple detection methods, which include pseudouridine-specific chemical derivatization and gas phase dissociation of RNA during liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of total tRNA isolated from E. coli pseudouridine synthase knock-out mutants identified RluF as the enzyme responsible for this modification. Furthermore, the absence of this modification compromises the translational ability of a luciferase reporter gene coding sequence when it is preceded by multiple tyrosine codons. This effect has implications for the translation of mRNAs that are rich in tyrosine codons in bacterial expression systems.

Importance of single molecular determinants in the fidelity of expanded genetic codes.

The site-selective encoding of noncanonical amino acids (NAAs) is a powerful technique for the installation of novel chemical functional groups in proteins. This is often achieved by recoding a stop codon and requires two additional components: an evolved aminoacyl tRNA synthetase (AARS) and a cognate tRNA. Analysis of the most successful AARSs reveals common characteristics. The highest fidelity NAA systems derived from the Methanocaldococcus jannaschii tyrosyl AARS feature specific mutations to two residues reported to interact with the hydroxyl group of the substrate tyrosine. We demonstrate that the restoration of just one of these determinants for amino acid specificity results in the loss of fidelity as the evolved AARSs become noticeably promiscuous. These results offer a partial explanation of a recently retracted strategy for the synthesis of glycoproteins. Similarly, we reinvestigated a tryptophanyl AARS reported to allow the site-selective incorporation of 5-hydroxy tryptophan within mammalian cells. In multiple experiments, the enzyme displayed elements of promiscuity despite its previous characterization as a high fidelity enzyme. Given the many similarities of the TyrRSs and TrpRSs reevaluated here, our findings can be largely combined, and in doing so they reinforce the long-established central dogma regarding the molecular basis by which these enzymes contribute to the fidelity of translation. Thus, our view is that the central claims of fidelity reported in several NAA systems remain unproven and unprecedented.

Exploring genetic codon expansion for unnatural amino acid incorporation in filamentous fungus Aspergillus nidulans.

Genetic code expansion technology allows the incorporation of unnatural amino acids (UAAs) into proteins, which is useful in protein engineering, synthetic biology, and gene therapy. Despite its potential applications in various species, filamentous fungi remain unexplored. This study aims to address this gap by developing these techniques in Aspergillus nidulans. We introduced an amber stop codon into a specific sequence within the reporter gene expressed in A. nidulans and replaced the anticodon of the fungal tRNATyr with CUA. This resulted in the synthesis of the target protein, confirming the occurrence of amber suppression in the fungus. When exogenous E. coli tRNATyrCUA (Ec. tRNATyrCUA) and E. coli tyrosyl-tRNA (Ec.TyrRS) were introduced into A. nidulans, they successfully synthesized the target protein via amber suppression and were shown to be orthogonal to the fungal translation system. By replacing the wild-type Ec.TyrRS with a mutant with a higher affinity for the UAA O-methyl-L-tyrosine, the fungal system was able to initiate the synthesis of the UAA-labeled protein (UAA-protein). We further increased the expression level of the UAA-protein through several rational modifications. The successful development of a genetic code expansion technique for A. nidulans has introduced a potentially valuable approach to the study of fungal protein structure and function.

Characterization of the human tRNA-guanine transglycosylase: confirmation of the heterodimeric subunit structure.

The eukaryotic tRNA-guanine transglycosylase (TGT) has been reported to exist as a heterodimer, in contrast to the homodimeric eubacterial TGT. While ubiquitin-specific protease 14 (USP14) has been proposed to act as a regulatory subunit of the eukaryotic TGT, the mouse TGT has recently been shown to be a queuine tRNA-ribosyltransferase 1 (QTRT1, eubacterial TGT homolog).queuine tRNA-ribosyltransferase domain-containing 1 (QTRTD1) heterodimer. We find that human QTRTD1 (hQTRTD1) co-purifies with polyhistidine-tagged human QTRT1 (ht-hQTRT1) via Ni(2+) affinity chromatography. Cross-linking experiments, mass spectrometry, and size exclusion chromatography results are consistent with the two proteins existing as a heterodimer. We have not been able to observe co-purification and/or association between hQTRT1 and USP14 when co-expressed in Escherichia coli. More importantly, under our experimental conditions, the transglycosylase activity of hQTRT1 is only observed when hQTRT1 and hQTRTD1 have been co-expressed and co-purified. Kinetic characterization of the human TGT (hQTRT1.hQTRTD1) using human tRNA(Tyr) and guanine shows catalytic efficiency (k(cat)/K(M)) similar to that of the E. coli TGT. Furthermore, site-directed mutagenesis confirms that the hQTRT1 subunit is responsible for the transglycosylase activity. Taken together, these results indicate that the human TGT is composed of a catalytic subunit, hQTRT1, and hQTRTD1, not USP14. hQTRTD1 has been implicated as the salvage enzyme that generates free queuine from QMP. Work is ongoing in our laboratory to confirm this activity.

Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion.

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase-tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS-tRNA(Tyr) pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNA(Tyr). The endogenous TyrRS and tRNA(Tyr) genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS-tRNA(Tyr) pair. In this engineered strain, 3-iodo-L-tyrosine and 3-azido-L-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-L-tyrosine and was also found to recognize 3-azido-L-tyrosine. The structural basis for the 3-azido-L-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.

Sensory ataxic neuropathy in golden retriever dogs is caused by a deletion in the mitochondrial tRNATyr gene.

Sensory ataxic neuropathy (SAN) is a recently identified neurological disorder in golden retrievers. Pedigree analysis revealed that all affected dogs belong to one maternal lineage, and a statistical analysis showed that the disorder has a mitochondrial origin. A one base pair deletion in the mitochondrial tRNA(Tyr) gene was identified at position 5304 in affected dogs after re-sequencing the complete mitochondrial genome of seven individuals. The deletion was not found among dogs representing 18 different breeds or in six wolves, ruling out this as a common polymorphism. The mutation could be traced back to a common ancestor of all affected dogs that lived in the 1970s. We used a quantitative oligonucleotide ligation assay to establish the degree of heteroplasmy in blood and tissue samples from affected dogs and controls. Affected dogs and their first to fourth degree relatives had 0-11% wild-type (wt) sequence, while more distant relatives ranged between 5% and 60% wt sequence and all unrelated golden retrievers had 100% wt sequence. Northern blot analysis showed that tRNA(Tyr) had a 10-fold lower steady-state level in affected dogs compared with controls. Four out of five affected dogs showed decreases in mitochondrial ATP production rates and respiratory chain enzyme activities together with morphological alterations in muscle tissue, resembling the changes reported in human mitochondrial pathology. Altogether, these results provide conclusive evidence that the deletion in the mitochondrial tRNA(Tyr) gene is the causative mutation for SAN.

Biochemical and genetic evidence for a role of IGHMBP2 in the translational machinery.

The human motor neuron degenerative disease spinal muscular atrophy with respiratory distress type 1 (SMARD1) is caused by loss of function mutations of immunoglobulin mu-binding protein 2 (IGHMBP2), a protein of unknown function that contains DNA/RNA helicase and nucleic acid-binding domains. Reduced IGHMBP2 protein levels in neuromuscular degeneration (nmd) mice, the mouse model of SMARD1, lead to motor neuron degeneration. We report the biochemical characterization of IGHMBP2 and the isolation of a modifier locus that rescues the phenotype and motor neuron degeneration of nmd mice. We find that a 166 kb BAC transgene derived from CAST/EiJ mice and containing tRNA genes and activator of basal transcription 1 (Abt1), a protein-coding gene that is required for ribosome biogenesis, contains the genetic modifier responsible for motor neuron rescue. Our biochemical investigations show that IGHMBP2 associates physically with tRNAs and in particular with tRNA(Tyr), which are present in the modifier and with the ABT1 protein. We find that transcription factor IIIC-220 kDa (TFIIIC220), an essential factor required for tRNA transcription, and the helicases Reptin and Pontin, which function in transcription and in ribosome biogenesis, are also part of IGHMBP2-containing complexes. Our findings strongly suggest that IGHMBP2 is a component of the translational machinery and that these components can be manipulated genetically to suppress motor neuron degeneration.

Targeted mRNA degradation by complex-mediated delivery of antisense RNAs to intracellular human mitochondria.

Mitochondrial dysfunction underlies a large number of acute or progressive diseases, as well as aging. However, proposed therapies for mitochondrial mutations suffer from poor transformation of mitochondria with exogenous DNA, or lack of functionality of the transferred nucleic acid within the organelle. We show that a transfer RNA import complex (RIC) from the parasitic protozoon Leishmania tropica rapidly and efficiently delivered signal-tagged antisense (STAS) RNA or DNA to mitochondria of cultured human cells. STAS-induced specific degradation of the targeted mitochondrial mRNA, with downstream effects on respiration. These results reveal the existence of a novel small RNA-mediated mRNA degradation pathway in mammalian mitochondria, and suggest that RIC-mediated delivery could be used to target therapeutic RNAs to the organelle within intact cells.