Cytosolic yeast tRNA(His) is covalently modified when imported into mitochondria of Trypanosoma brucei.

The mitochondrial genome of Trypanosoma brucei does not encode any tRNAs. Instead, mitochondrial tRNAs are synthesized in the nucleus and subsequently imported into mitochondria. The great majority of mitochondrial tRNAs have cytosolic counterparts showing identical primary sequences. The only difference found between mitochondrial and cytosolic isotypes of the tRNAs are mitochondria-specific nucleotide modifications which appear to be a common feature of imported tRNAs in trypanosomes. In this study, a mutated yeast cytosolic tRNAHis was expressed in trypanosomes and its import phenotype was analyzed by cell fractionation and nuclease treatment of intact mitochondria. Furthermore, cytosolic and mitochondrial isotypes of the yeast tRNA(His) were specifically labeled and analyzed by limited alkaline hydrolysis. These experiments revealed the presence of mitochondria-specific nucleotide modifications in the yeast tRNA(His). The positions of the modifications were determined by direct enzymatic sequencing of the tRNA(His) and shown to correspond to the ultimate and penultimate nucleotides before the anticodon, the same relative positions which are modified in the mitochondrial isotype of trypanosomal tRNA(Tyr). The results demonstrate that covalent modification of tRNAs; in trypanosomal mitochondria can be used, in analogy to processing of precursor proteins during mitochondrial protein import, as a marker for import of both endogenous and heterologous tRNAs.     

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.     

Another cut for lysine tRNA: application of the hyperprocessing reaction reveals another stabilization strategy in metazoan lysine tRNAs

Recently, we revealed that the cloverleaf structure of some eukaryotic tRNAs is not always stable in vitro, and the denatured structures of these tRNAs are sometimes detected in bacterial RNase P reactions. We have designated the unusual internal cleavage reaction of these tRNAs as hyperprocessing. We have developed this hyperprocessing strategy as a useful tool for examining the stability of the tRNA cloverleaf structure. There are some common features in such unstable, hyperprocessible tRNAs, and the criteria for the hyperprocessing reaction of tRNA are extracted. Metazoan initiator methionine tRNAs and lysine tRNAs commonly fit the criteria, and are predicted to be hyperprocessible. The RNase P reactions of two metazoan lysine tRNAs from Homo sapiens and Caenorhabditis elegans, which fit the criteria, resulted in resistance to the internal cleavage reaction, while one bacterial lysine tRNA from Acholeplasma laidlawii, which also fits the criteria, was internally cleaved by the RNase P. The results showed that the metazoan lysine tRNAs examined are very stable without base modifications even under in vitro conditions. We also examined the 3'-half short construct of the human lysine tRNA, and the results showed that this RNA was internally cleaved by the enzyme. The results indicated that the human lysine tRNA has the ability to be hyperprocessed but is structurally stabilized in spite of lacking base modifications. A comparative study suggested, moreover, that the acceptor-stem bases should take part in the stabilization of metazoan lysine tRNAs. Our data strongly suggest that the cloverleaf shape of other metazoan lysine tRNAs should also be stabilized by means of similar strategies to in the case of human tRNA(Lys3).     

The T-stem determines the cytosolic or mitochondrial localization of trypanosomal tRNAsMet

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Organellar translation therefore depends on import of cytosolic, nucleus-encoded tRNAs. Except for the cytosol-specific initiator tRNA(Met), all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The initiator tRNA(Met) is closely related to the imported elongator tRNA(Met). Thus, the distinct localization of the two tRNAs(Met) must be specified by the 26 nucleotides, which differ between the two molecules. Using transgenic T. brucei cell lines and subsequent cell fractionation, we show that the T-stem is both required and sufficient to specify the localization of the tRNAs(Met). Furthermore, it was shown that the tRNA(Met) T-stem localization determinants are also functional in the context of two other tRNAs. In vivo analysis of the modified nucleotides found in the initiator tRNA(Met) indicates that the T-stem localization determinants do not require modified nucleotides. In contrast, import of native tRNAs(Met) into isolated mitochondria suggests that nucleotide modifications might be involved in regulating the extent of import of elongator tRNA(Met).     

Identification of yeast tRNA Um(44) 2'-O-methyltransferase (Trm44) and demonstration of a Trm44 role in sustaining levels of specific tRNA(Ser) species

A characteristic feature of tRNAs is the numerous modifications found throughout their sequences, which are highly conserved and often have important roles. Um(44) is highly conserved among eukaryotic cytoplasmic tRNAs with a long variable loop and unique to tRNA(Ser) in yeast. We show here that the yeast ORF YPL030w (now named TRM44) encodes tRNA(Ser) Um(44) 2'-O-methyltransferase. Trm44 was identified by screening a yeast genomic library of affinity purified proteins for activity and verified by showing that a trm44-delta strain lacks 2'-O-methyltransferase activity and has undetectable levels of Um(44) in its tRNA(Ser) and by showing that Trm44 purified from Escherichia coli 2'-O-methylates U(44) of tRNA(Ser) in vitro. Trm44 is conserved among metazoans and fungi, consistent with the conservation of Um(44) in eukaryotic tRNAs, but surprisingly, Trm44 is not found in plants. Although trm44-delta mutants have no detectable growth defect, TRM44 is required for survival at 33 degrees C in a tan1-delta mutant strain, which lacks ac(4)C12 in tRNA(Ser) and tRNA(Leu). At nonpermissive temperature, a trm44-delta tan1-delta mutant strain has reduced levels of tRNA(Ser(CGA)) and tRNA(Ser(UGA)), but not other tRNA(Ser) or tRNA(Leu) species. The trm44-delta tan1-delta growth defect is suppressed by addition of multiple copies of tRNA(Ser(CGA)) and tRNA(Ser(UGA)), directly implicating these tRNA(Ser) species in this phenotype. The reduction of specific tRNA(Ser) species in a trm44-delta tan1-delta mutant underscores the importance of tRNA modifications in sustaining tRNA levels and further emphasizes that tRNAs undergo quality control.     

Changes in transfer ribonucleic acids of Bacillus subtilis during different growth phases

The transfer ribonucleic acids (tRNAs) of B. subtilis at different growth phases are examined for changes in the composition and the methylation of minor constituents. The composition of the tRNAs indicates about equal amounts of adenosine and uridine, and of guanosine and cytidine. About 3-4 residues are present as modified bases in the average tRNA molecule. The net composition of tRNAs appears to remain unaltered during different growth phases. In vitro methylation of tRNAs indicates lack of methyl groups in both exponentially growing cells and spores. In vivo methylation studies show tRNA methylation occurs during the stationary phase in the absence of net tRNA synthesis. Thus, both in vitro and in vivo methylation indicates that the tRNAs in exponentially growing cells do not contain their full complement of modified bases. More complete modification is noted in tRNAs from stationary cells or spores. Hence, tRNA modifications in general are preserved with fidelity even in the dormant spore but the possibility is left open that specific modifications of selected isoacceptors of tRNAs may occur.     

28 The study of tRNA modifications by molecular dynamics

Modified nucleosides are prevalent in tRNA. Experimental studies reveal that modifications play an important role in tuning tRNA activity. In this study, molecular dynamics (MD) simulations were used to investigate how modifications alter tRNA structure and dynamics. The X-ray crystal structures of tRNA-Asp, tRNA-Phe, and tRNA-iMet, both with and without modifications, were used as initial structures for 333-ns time-scale MD trajectories with AMBER. For each tRNA molecule, three independent trajectory calculations were performed. Force field parameters were built using the RESP procedure of Cieplak et al. for 17 nonstandard tRNA residues. The global root-mean-square deviations (RMSDs) of atomic positions show that modifications only introduce significant rigidity to tRNA-Phe’s global structure. Interestingly, regional RMSDs of anticodon stem-loop suggest that modified tRNA has more rigid structure compared to the unmodified tRNA in this domain. The anticodon RMSDs of the modified tRNAs, however, are higher than those of corresponding unmodified tRNAs. These findings suggest that rigidity of the anticodon arm is essential for tRNA translocation in the ribosome complex, and, on the other hand, flexibility of anticodon might be critical for anticodon–codon recognition. We also measure the angle between the 3D L-shaped arms of tRNA; backbone atoms of acceptor stem and TψC stem loop are selected to indicate one vector, and backbone atoms of anticodon stem and D stem loop are selected to indicate the other vector. By measuring the angle between two vectors, we find that the initiator tRNA has a narrower range of hinge motion compared to tRNA-Asp and tRNA-Phe, which are elongator tRNA. This suggests that elongator tRNAs, which might require significant flexibility in this hinge to transition from the A–to-P site in the ribosome, have evolved to specifically accommodate this need.     

Structure and function of initiator methionine tRNA from the mitochondria of Neurospora crassa.

Initiator methionine tRNA from the mitochondria of Neurospora crassa has been purified and sequenced. This mitochondrial tRNA can be aminoacylated and formylated by E. coli enzymes, and is capable of initiating protein synthesis in E. coli extracts. The nucleotide composition of the mitochondrial initiator tRNA (the first mitochondrial tRNA subjected to sequence analysis) is very rich in A + U, like that reported for total mitochondrial tRNA. In two of the unique features which differentiate procaryotic from eucaryotic cytoplasmic initiator tRNAs, the mitochondrial tRNA appears to resemble the eucaryotic initiator tRNAs. Thus unlike procaryotic initiator tRNAs in which the 5′ terminal nucleotide cannot form a Watson-Crick base pair to the fifth nucleotide from the 3′ end, the mitochondrial tRNA can form such a base pair; and like the eucaryotic cytoplasmic initiator tRNAs, the mitochondrial initiator tRNA lacks the sequence -TΨCG(or A) in loop IV. The corresponding sequence in the mitochondrial tRNA, however, is -UGCA- and not -AU(or Ψ)CG-as found in all eucaryotic cytoplasmic initiator tRNAs. In spite of some similarity of the mitochondrial initiator tRNA to both eucaryotic and procaryotic initiator tRNAs, the mitochondrial initiator tRNA is basically different from both these tRNAs. Between these two classes of initiator tRNAs, however, it is more homologous in sequence to procaryotic (56–60%) than to eucaryotic cytoplasmic initiator tRNAs (45–51%).     

In vitro hyperprocessing of Drosophila tRNAs by the catalytic RNA of RNase P the cloverleaf structure of tRNA is not always stable?

We have previously reported that the catalytic RNA subunit of RNase P of Escherichia coli (M1 RNA) cleaves Drosophila initiator methionine tRNA (tRNA(Met)i) within the mature tRNA sequence to produce specific fragments. This cleavage was dependent on the occurrence of an altered conformation of the tRNA substrate. We call this further cleavage hyperprocessing. In the present paper, to search for another tRNA that can be hyperprocessed in vitro, we used total mature tRNAs from Drosophila as substrates for the in vitro M1 RNA reaction. We found that some tRNAs can be hyperprocessed by M1 RNA and that two such tRNAs are an alanine tRNA and a histidine tRNA. Using mutant substrates of these tRNAs, we also show that the hyperprocessing by M1 RNA is dependent on the occurrence of altered conformations of these tRNAs. The altered conformations were very similar to that of tRNA(Met)i. We show here that M1 RNA can be used as a powerful tool to detect the alternative conformation of tRNAs. The relationship between these hyperprocessing reactions and stability of the tRNA structure will also be discussed.     

Wobble modification deficiency in mutant tRNAs in patients with mitochondrial diseases

Point mutations in mitochondrial (mt) tRNA genes are associated with a variety of human mitochondrial diseases. We have shown previously that mt tRNA(Leu(UUR)) with a MELAS A3243G mutation and mt tRNA(Lys) with a MERRF A8344G mutation derived from HeLa background cybrid cells are deficient in normal taurine-containing modifications [taum(5)(s(2))U; 5-taurinomethyl-(2-thio)uridine] at the anticodon wobble position in both cases. The wobble modification deficiency results in defective translation. We report here wobble modification deficiencies of mutant mt tRNAs from cybrid cells with different nuclear backgrounds, as well as from patient tissues. These findings demonstrate the generality of the wobble modification deficiency in mutant tRNAs in MELAS and MERRF.     

Loss of a Conserved tRNA Anticodon Modification Perturbs Plant Immunity

tRNA is the most highly modified class of RNA species, and modifications are found in tRNAs from all organisms that have been examined. Despite their vastly different chemical structures and their presence in different tRNAs, occurring in different locations in tRNA, the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent discoveries have revealed unprecedented complexity in the modification patterns of tRNA, their regulation and function, suggesting that each modified nucleoside in tRNA may have its own specific function. However, in plants, our knowledge on the role of individual tRNA modifications and how they are regulated is very limited. In a genetic screen designed to identify factors regulating disease resistance and activation of defenses in Arabidopsis, we identified SUPPRESSOR OF CSB3 9 (SCS9). Our results reveal SCS9 encodes a tRNA methyltransferase that mediates the 2´-O-ribose methylation of selected tRNA species in the anticodon loop. These SCS9-mediated tRNA modifications enhance during the course of infection with the bacterial pathogen Pseudomonas syringae DC3000, and lack of such tRNA modification, as observed in scs9 mutants, severely compromise plant immunity against the same pathogen without affecting the salicylic acid (SA) signaling pathway which regulates plant immune responses. Our results support a model that gives importance to the control of certain tRNA modifications for mounting an effective immune response in Arabidopsis, and therefore expands the repertoire of molecular components essential for an efficient disease resistance response.     

Role of ribothymidine in the thermal stability of transfer RNA as monitored by proton magnetic resonance.

In order to elucidate the functional role of the modified uridines at position 54 of tRNA, the 270 MHz high-field proton NMR spectra of methionine tRNAs from E. coli, from a mutant thereof, and from T. thermophilus, containing ribothymidine, uridine and 2-thioribothymidine, respectively, have been measured as a function of temperature. A comparison of the NMR melting profiles of the minor nucleosides from these tRNAs shows that the melting temperature of uridine containing tRNA is 6 degrees C lower than that of the wild type tRNA whereas that of the 2-thioribothymidine tRNA is 7 degrees C higher than that of the wild type tRNA. These results, therefore, demonstrate that these modifications serve for stabilization of the tertiary structure of tRNA.