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.

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.

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.

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.

Role of disordered regions in transferring tyrosine to its cognate tRNA.

Aminoacyl tRNA synthetase (AARS) plays an important role in transferring each amino acid to its cognate tRNA. Specifically, tyrosyl tRNA synthetase (TyrRS) is involved in various functions including protection from DNA damage due to oxidative stress, protein synthesis and cell signaling and can be an attractive target for controlling the pathogens by early inhibition of translation. TyrRS has two disordered regions, which lack a stable 3D structure in solution, and are involved in tRNA synthetase catalysis and stability. One of the disordered regions undergoes disorder-to-order transition (DOT) upon complex formation with tRNA whereas the other remains disordered (DR). In this work, we have explored the importance of these disordered regions using molecular dynamics simulations of both free and RNA-complexed states. We observed that the DOT and DR regions of the first subunit acts as a flap and interact with the acceptor arm of the tRNA. The DOT-DR flap closes when tyrosine (TyrRSTyr) is present at the active site of the complex and opens in the presence of tyrosine monophosphate (TyrRSYMP). The DOT and DR regions of the second subunit interact with the anticodon stem as well as D-loop of the tRNA, which might be involved in stabilizing the complex. The anticodon loop of the tRNA binds to the structured region present in the C-terminal of the protein, which is observed to be flexible during simulations. Detailed energy calculations also show that TyrRSTyr complex has stronger binding energy between tRNA and protein compared to TyrRSYMP; on the contrary, the anticodon is strongly bound in TyrRSYMP. The results obtained in the present study provide additional insights for understanding catalysis and the involvement of disordered regions in Tyr transfer to cognate tRNA.

Plasmodium apicoplast tyrosyl-tRNA synthetase recognizes an unusual, simplified identity set in cognate tRNATyr.

The life cycle of Plasmodium falciparum, the agent responsible for malaria, depends on both cytosolic and apicoplast translation fidelity. Apicoplast aminoacyl-tRNA synthetases (aaRS) are bacterial-like enzymes devoted to organellar tRNA aminoacylation. They are all encoded by the nuclear genome and are translocated into the apicoplast only after cytosolic biosynthesis. Apicoplast aaRSs contain numerous idiosyncratic sequence insertions: An understanding of the roles of these insertions has remained elusive and they hinder efforts to heterologously overexpress these proteins. Moreover, the A/T rich content of the Plasmodium genome leads to A/U rich apicoplast tRNA substrates that display structural plasticity. Here, we focus on the P. falciparum apicoplast tyrosyl-tRNA synthetase (Pf-apiTyrRS) and its cognate tRNATyr substrate (Pf-apitRNATyr). Cloning and expression strategies used to obtain an active and functional recombinant Pf-apiTyrRS are reported. Functional analyses established that only three weak identity elements in the apitRNATyr promote specific recognition by the cognate Pf-apiTyrRS and that positive identity elements usually found in the tRNATyr acceptor stem are excluded from this set. This finding brings to light an unusual behavior for a tRNATyr aminoacylation system and suggests that Pf-apiTyrRS uses primarily negative recognition elements to direct tyrosylation specificity.

The pseudo-dimeric tyrosyl-tRNA synthetase of T. brucei aminoacylates cytosolic and mitochondrial tRNATyr and requires both monomeric units for activity.

Aminoacyl-tRNA synthetases are essential for protein synthesis. The single-copy tyrosyl-tRNA synthetase (Tb-TyrRS) of T. brucei has an unusual structure and forms a pseudo-dimer. It is therefore twice the size than tyrosyl-tRNA synthetases of most other organisms. Here we show by inducible RNAi that Tb-TyrRS is essential for normal growth of procyclic T. brucei. Furthermore we demonstrate that Tb-TyrRS aminoacylates cytosolic as well as mitochondrial tRNATyr indicating that it is dually localized. Finally we show that individual deletion of the 36 N- or C-terminal amino acids abolishes the function of Tb-TyrRS. This indicates that both monomeric units of the enzyme, the C-terminal one of which is predicted to lack enzymatic activity, are essential for Tb-TyrRS function. In summary our results together with previous studies support the notion that Tb-TyrRS might be a suitable drug target.

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.

Long and branched polyamines are required for maintenance of the ribosome, tRNAHis and tRNATyr in Thermus thermophilus cells at high temperatures.

Thermus thermophilus is an extremely thermophilic eubacterium that produces various polyamines. Aminopropylagmatine ureohydrolase (SpeB) and SAM decarboxylase-like protein 1 (SpeD1) are involved in the biosynthesis of spermidine from arginine. Because long and branched polyamines in T. thermophilus are synthesized from spermidine, the speB and speD1 gene-deleted strains (ΔspeB and ΔspeD1, respectively) cannot synthesize long and branched polyamines. Although neither strain grew at high temperatures (>75°C) in minimal medium, both strains survived at 80°C when they were cultured at 70°C until the mid-log phase and then shifted to 80°C. We therefore prepared the ΔspeB and ΔspeD1 cells using this culture method. Microscopic analysis showed that both strains can survive for 10 h after the temperature shift. Although the modification levels of 2'-O-methylguanosine at position 18, N7 -methylguanosine at position 46, 5-methyluridine at position 54 and N1 -methyladenosine at position 58 in the class I tRNA from both strains were normal, amounts of tRNATyr , tRNAHis , rRNAs and 70S ribosomes were decreased after the temperature shift. Furthermore, in vivo protein synthesis in both strains was completely lost 10 h after the temperature shift. Thus, long and branched polyamines are required for at least the maintenance of 70S ribosome and some tRNA species at high temperatures.

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.

The essential function of the Trypanosoma brucei Trl1 homolog in procyclic cells is maturation of the intron-containing tRNATyr.

Trypanosoma brucei, the etiologic agent of sleeping sickness, encodes a single intron-containing tRNA, tRNA(Tyr), and splicing is essential for its viability. In Archaea and Eukarya, tRNA splicing requires a series of enzymatic steps that begin with intron cleavage by a tRNA-splicing endonuclease and culminates with joining the resulting tRNA exons by a splicing tRNA ligase. Here we explored the function of TbTrl1, the T. brucei homolog of the yeast Trl1 tRNA ligase. We used a combination of RNA interference and molecular biology approaches to show that down-regulation of TbTrl1 expression leads to accumulation of intron-containing tRNA(Tyr) and a concomitant growth arrest at the G1 phase. These defects were efficiently rescued by expression of an "intronless" version of tRNA(Tyr) in the same RNAi cell line. Taken together, these experiments highlight the crucial importance of the TbTrl1 for tRNA(Tyr) maturation and viability, while revealing tRNA splicing as its only essential function.

Lack of tRNA-i6A modification causes mitochondrial-like metabolic deficiency in S. pombe by limiting activity of cytosolic tRNATyr, not mito-tRNA.

tRNA-isopentenyl transferases (IPTases) are highly conserved enzymes that form isopentenyl-N(6)-A37 (i6A37) on subsets of tRNAs, enhancing their translation activity. Nuclear-encoded IPTases modify select cytosolic (cy-) and mitochondrial (mt-) tRNAs. Mutation in human IPTase, TRIT1, causes disease phenotypes characteristic of mitochondrial translation deficiency due to mt-tRNA dysfunction. Deletion of the Schizosaccharomyces pombe IPTase (tit1-Δ) causes slow growth in glycerol, as well as in rapamycin, an inhibitor of TOR kinase that maintains metabolic homeostasis. Schizosaccharomyces pombe IPTase modifies three different cy-tRNAs(Ser) as well as cy-tRNA(Tyr), cy-tRNA(Trp), and mt-tRNA(Trp). We show that lower ATP levels in tit1-Δ relative to tit1(+) cells are also more decreased by an inhibitor of oxidative phosphorylation, indicative of mitochondrial dysfunction. Here we asked if the tit1-Δ phenotypes are due to hypomodification of cy-tRNA or mt-tRNA. A cytosol-specific IPTase that modifies cy-tRNA, but not mt-tRNA, fully rescues the tit1-Δ phenotypes. Moreover, overexpression of cy-tRNAs also rescues the phenotypes, and cy-tRNA(Tyr) alone substantially does so. Bioinformatics indicate that cy-tRNA(Tyr) is most limiting for codon demand in tit1-Δ cells and that the cytosolic mRNAs most loaded with Tyr codons encode carbon metabolilizing enzymes, many of which are known to localize to mitochondria. Thus, S. pombe i6A37 hypomodification-associated metabolic deficiency results from hypoactivity of cy-tRNA, mostly tRNA(Tyr), and unlike human TRIT1-deficiency does not impair mitochondrial translation due to mt-tRNA hypomodification. We discuss species-specific aspects of i6A37. Specifically relevant to mitochondria, we show that its hypermodified version, ms2i6A37 (2-methylthiolated), which occurs on certain mammalian mt-tRNAs (but not cy-tRNAs), is not found in yeast.

Mitochondrial poly(A) polymerase is involved in tRNA repair.

Transcription of the mitochondrial genome results in polycistronic precursors, which are processed mainly by the release of tRNAs interspersed between rRNAs and mRNAs. In many metazoan mitochondrial genomes some tRNA genes overlap with downstream genes; in the case of human mitochondria the genes for tRNA(Tyr) and tRNA(Cys) overlap by one nucleotide. It has previously been shown that processing of the common precursor releases an incomplete tRNA(Tyr) lacking the 3'-adenosine. The 3'-terminal adenosine has to be added before addition of the CCA end and subsequent aminoacylation. We show that the mitochondrial poly(A) polymerase (mtPAP) is responsible for this A addition. In vitro, a tRNA(Tyr) lacking the discriminator is a substrate for mtPAP. In vivo, an altered mtPAP protein level affected tRNA(Tyr) maturation, as shown by sequencing the 3' ends of mitochondrial tRNAs. Complete repair could be reconstituted in vitro with three enzymes: mtPAP frequently added more than one A to the 3' end of the truncated tRNA, and either the mitochondrial deadenylase PDE12 or the endonuclease RNase Z trimmed the oligo(A) tail to a single A before CCA addition. An enzyme machinery that evolved primarily for other purposes thus allows to tolerate the frequent evolutionary occurrence of gene overlaps.

Studies of translational misreading in vivo show that the ribosome very efficiently discriminates against most potential errors.

Protein synthesis must rapidly and repeatedly discriminate between a single correct and many incorrect aminoacyl-tRNAs. We have attempted to measure the frequencies of all possible missense errors by tRNA , tRNA and tRNA . The most frequent errors involve three types of mismatched nucleotide pairs, U•U, U•C, or U•G, all of which can form a noncanonical base pair with geometry similar to that of the canonical U•A or C•G Watson-Crick pairs. Our system is sensitive enough to measure errors at other potential mismatches that occur at frequencies as low as 1 in 500,000 codons. The ribosome appears to discriminate this efficiently against any pair with non-Watson-Crick geometry. This extreme accuracy may be necessary to allow discrimination against the errors involving near Watson-Crick pairing.

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.

Pleiotropic effects of a mitochondrial-nuclear incompatibility depend upon the accelerating effect of temperature in Drosophila.

Interactions between mitochondrial and nuclear gene products that underlie eukaryotic energy metabolism can cause the fitness effects of mutations in one genome to be conditional on variation in the other genome. In ectotherms, the effects of these interactions are likely to depend upon the thermal environment, because increasing temperature accelerates molecular rates. We find that temperature strongly modifies the pleiotropic phenotypic effects of an incompatible interaction between a Drosophila melanogaster polymorphism in the nuclear-encoded, mitochondrial tyrosyl-transfer (t)RNA synthetase and a D. simulans polymorphism in the mitochondrially encoded tRNA(Tyr). The incompatible mitochondrial-nuclear genotype extends development time, decreases larval survivorship, and reduces pupation height, indicative of decreased energetic performance. These deleterious effects are ameliorated when larvae develop at 16° and exacerbated at warmer temperatures, leading to complete sterility in both sexes at 28°. The incompatible genotype has a normal metabolic rate at 16° but a significantly elevated rate at 25°, consistent with the hypothesis that inefficient energy metabolism extends development in this genotype at warmer temperatures. Furthermore, the incompatibility decreases metabolic plasticity of larvae developed at 16°, indicating that cooler development temperatures do not completely mitigate the deleterious effects of this genetic interaction. Our results suggest that the epistatic fitness effects of metabolic mutations may generally be conditional on the thermal environment. The expression of epistatic interactions in some environments, but not others, weakens the efficacy of selection in removing deleterious epistatic variants from populations and may promote the accumulation of incompatibilities whose fitness effects will depend upon the environment in which hybrids occur.