Pathology-related substitutions in human mitochondrial tRNA(Ile) reduce precursor 3' end processing efficiency in vitro

The human mitochondrial genome encodes 22 tRNAs interspersed among the two rRNAs and 11 mRNAs, often without spacers, suggesting that tRNAs must be efficiently excised. Numerous maternally transmitted diseases and syndromes arise from mutations in mitochondrial tRNAs, likely due to defect(s) in tRNA metabolism. We have systematically explored the effect of pathogenic mutations on tRNA^Ile precursor 3′ end maturation in vitro by 3′-tRNase. Strikingly, four pathogenic tRNA^Ile mutations reduce 3′-tRNase processing efficiency (V_max / K_M) to ~10-fold below that of wild-type, principally due to lower V_max. The structural impact of mutations was sought by secondary structure probing and wild-type tRNA^Ile precursor was found to fold into a canonical cloverleaf. Among the mutant tRNA^Ile precursors with the greatest 3′ end processing deficiencies, only G4309A displays a secondary structure substantially different from wild-type, with changes in the T domain proximal to the substitution. Reduced efficiency of tRNA^Ile precursor 3′ end processing, in one case associated with structural perturbations, could thus contribute to human mitochondrial diseases caused by mutant tRNAs.     

Asymmetric maturation of a dimeric transfer RNA precursor

Six of the eight transfer RNAs coded by bacteriophage T4 are synthesized via three dimeric precursor molecules. The sequences of two of these have been determined. Both of these precursors give rise to equimolar amounts of the cognate tRNA molecules in vivo. In contrast, even in wild-type infections, tRNA^Ile is present in ≤ 30% the amount of tRNA^Thr, with which it is processed from a common dimeric precursor.We have now determined the sequence of this dimer. In addition to the nucleotides present in tRNA^Thr and tRNA^Ile, it contains nine precursor-specific residues, located at the 5′ and 3′ termini and at the interstitial junction of the two tRNA sequences. While the three dimers share the majority of structural features in common, pre-tRNA^{Thr + Ile} is the only case in which an encoded tRNA 3′ -C-C-A terminus is present in the interstitial region.The processing of this dimer in various biosynthetic mutants has been analyzed in vivo and in vitro and shown to be anomalous in several respects. These results suggest that the apparent underproduction of tRNA^Ile can be explained by a novel processing pathway that generates a metabolically unstable tRNA^Ile product. Data from DNA sequence analysis of the T4 tRNA gene cluster (Fukada & Abelson, 1980) support the conclusion that the asymmetric maturation of this precursor is a consequence of the unique disposition of the -C-C-A sequence. These results argue that gene expression can be modulated at the level of RNA processing. The biological significance of this phenomenon is discussed in relation to evidence that tRNA^Ile has a unique physiological role.     

Mechanistic insights into mitochondrial tRNAAla 3’-end metabolism deficiency

Mitochondrial tRNA 3’-end metabolism is critical for the formation of functional tRNAs. Deficient mitochondrial tRNA 3’-end metabolism is linked to an array of human diseases, including optic neuropathy, but their pathophysiology remains poorly understood. In this report, we investigated the molecular mechanism underlying the Leber’s hereditary optic neuropathy (LHON)-associated tRNA^Ala 5587A>G mutation, which changes a highly conserved adenosine at position 73 (A73) to guanine (G73) on the 3’-end of the tRNA acceptor stem. The m.5587A>G mutation was identified in three Han Chinese families with suggested maternal inheritance of LHON. We hypothesized that the m.5587A>G mutation altered tRNA^Ala 3’-end metabolism and mitochondrial function. In vitro processing experiments showed that the m.5587A>G mutation impaired the 3’-end processing of tRNA^Ala precursors by RNase Z and inhibited the addition of CCA by tRNA nucleotidyltransferase (TRNT1). Northern blot analysis revealed that the m.5587A>G mutation perturbed tRNA^Ala aminoacylation, as evidenced by decreased efficiency of aminoacylation and faster electrophoretic mobility of mutated tRNA^Ala in these cells. The impact of m.5587A>G mutation on tRNA^Ala function was further supported by increased melting temperature, conformational changes, and reduced levels of this tRNA. Failures in tRNA^Ala metabolism impaired mitochondrial translation, perturbed assembly and activity of oxidative phosphorylation complexes, diminished ATP production and membrane potential, and increased production of reactive oxygen species. These pleiotropic defects elevated apoptotic cell death and promoted mitophagy in cells carrying the m.5587A>G mutation, thereby contributing to visual impairment. Our findings may provide new insights into the pathophysiology of LHON arising from mitochondrial tRNA 3’-end metabolism deficiency.     

Arabidopsis Encodes Four tRNase Z Enzymes

Functional transfer RNA (tRNA) molecules are a prerequisite for protein biosynthesis. Several processing steps are required to generate the mature functional tRNA from precursor molecules. Two of the early processing steps involve cleavage at the tRNA 5′ end and the tRNA 3′ end. While processing at the tRNA 5′ end is performed by RNase P, cleavage at the 3′ end is catalyzed by the endonuclease tRNase Z. In eukaryotes, tRNase Z enzymes are found in two versions: a short form of about 250 to 300 amino acids and a long form of about 700 to 900 amino acids. All eukaryotic genomes analyzed to date encode at least one long tRNase Z protein. Of those, Arabidopsis (Arabidopsis thaliana) is the only organism that encodes four tRNase Z proteins, two short forms and two long forms. We show here that the four proteins are distributed to different subcellular compartments in the plant cell: the nucleus, the cytoplasm, the mitochondrion, and the chloroplast. One tRNase Z is present only in the cytoplasm, one protein is found exclusively in mitochondria, while the third one has dual locations: nucleus and mitochondria. None of these three tRNase Z proteins is essential. The fourth tRNase Z protein is present in chloroplasts, and deletion of its gene results in an embryo-lethal phenotype. In vitro analysis with the recombinant proteins showed that all four tRNase Z enzymes have tRNA 3′ processing activity. In addition, the mitochondrial tRNase Z proteins cleave tRNA-like elements that serve as processing signals in mitochondrial mRNA maturation.     

Naturally occurring mutations in human mitochondrial pre-tRNASer(UCN) can affect the transfer ribonuclease Z cleavage site, processing kinetics, and substrate secondary structure

tRNAs are transcribed as precursors with a 5' end leader and a 3' end trailer. The 5' end leader is processed by RNase P, and in most organisms in all three kingdoms, transfer ribonuclease (tRNase) Z can endonucleolytically remove the 3' end trailer. Long ((L)) and short ((S)) forms of the tRNase Z gene are present in the human genome. tRNase Z(L) processes a nuclear-encoded pre-tRNA approximately 1600-fold more efficiently than tRNase Z(S) and is predicted to have a strong mitochondrial transport signal. tRNase Z(L) could, thus, process both nuclear- and mitochondrially encoded pre-tRNAs. More than 150 pathogenesis-associated mutations have been found in the mitochondrial genome, most of them in the 22 mitochondrially encoded tRNAs. All the mutations investigated in human mitochondrial tRNA(Ser(UCN)) affect processing efficiency, and some affect the cleavage site and secondary structure. These changes could affect tRNase Z processing of mutant pre-tRNAs, perhaps contributing to mitochondrial disease.     

Molecular Characterization of Three PRORP Proteins in the Moss Physcomitrella patens: Nuclear PRORP Protein Is Not Essential for Moss Viability

RNase P is a ubiquitous endonuclease that removes the 5′ leader sequence from pre-tRNAs in all organisms. In Arabidopsis thaliana, RNA-free proteinaceous RNase Ps (PRORPs) seem to be enzyme(s) for pre-tRNA 5′-end processing in organelles and the nucleus and are thought to have replaced the ribonucleoprotein RNase P variant. However, the evolution and function of plant PRORPs are not fully understood. Here, we identified and characterized three PRORP-like proteins, PpPPR_63, 67, and 104, in the basal land plant, the moss Physcomitrella patens. PpPPR_63 localizes to the nucleus, while PpPPR_67 and PpPPR_104 are found in both the mitochondria and chloroplasts. The three proteins displayed pre-tRNA 5′-end processing activity in vitro. Mutants with knockout (KO) of the PpPPR_63 gene displayed growth retardation of protonemal colonies, indicating that, unlike Arabidopsis nuclear RPORPs, the moss nuclear PpPPR_63 is not essential for viability. In the KO mutant, nuclear-encoded tRNA^Asp (GUC) levels were slightly decreased, whereas most nuclear-encoded tRNA levels were not altered. This indicated that most of the cytosolic mature tRNAs were produced normally without proteinaceous RNase P-like PpPPR_63. Single PpPPR_67 or 104 gene KO mutants displayed different phenotypes of protonemal growth and chloroplast tRNA^Arg (ACG) accumulation. However, the levels of all other tRNAs were not altered in the KO mutants. In addition, in vitro RNase P assays showed that PpPPR_67 and PpPPR_104 efficiently cleaved chloroplast pre-tRNA^Arg (CCG) and pre-tRNA^Arg (UCU) but they cleaved pre-tRNA^Arg (ACG) with different efficiency. This suggests that the two proteins have overlapping function but their substrate specificity is not identical.     

Kinetic Proofreading at Single Molecular Level: Aminoacylation of tRNAIle and the Role of Water as an Editor

Proofreading/editing in protein synthesis is essential for accurate translation of information from the genetic code. In this article we present a theoretical investigation of efficiency of a kinetic proofreading mechanism that employs hydrolysis of the wrong substrate as the discriminatory step in enzyme catalytic reactions. We consider aminoacylation of tRNA^Ile which is a crucial step in protein synthesis and for which experimental results are now available. We present an augmented kinetic scheme and then employ methods of stochastic simulation algorithm to obtain time dependent concentrations of different substances involved in the reaction and their rates of formation. We obtain the rates of product formation and ATP hydrolysis for both correct and wrong substrates (isoleucine and valine in our case, respectively), in single molecular enzyme as well as ensemble enzyme kinetics. The present theoretical scheme correctly reproduces (i) the amplitude of the discrimination factor in the overall rates between isoleucine and valine which is obtained as (1.8×10²).(4.33×10²) = 7.8×10⁴, (ii) the rates of ATP hydrolysis for both Ile and Val at different substrate concentrations in the aminoacylation of tRNA^Ile. The present study shows a non-michaelis type dependence of rate of reaction on tRNA^Ile concentration in case of valine. The overall editing in steady state is found to be independent of amino acid concentration. Interestingly, the computed ATP hydrolysis rate for valine at high substrate concentration is same as the rate of formation of Ile-tRNA^Ile whereas at intermediate substrate concentration the ATP hydrolysis rate is relatively low. We find that the presence of additional editing domain in class I editing enzyme makes the kinetic proofreading more efficient through enhanced hydrolysis of wrong product at the editing CP1 domain.     

Modified nucleosides and the chromatographic and aminoacylation behavior of tRNAIle from Escherichia coli C6

Transfer RNA from Escherichia coli C6, a Met⁻, Cys⁻, relA⁻ mutant, was previously shown to contain an altered tRNA^Ile which accumulates during cysteine starvation (Harris, C.L., Lui, L., Sakallah, S. and DeVore, R. (1983) J. Biol. Chem. 258, 7676–7683). We now report the purification of this altered tRNA^Ile and a comparison of its aminoacylation and chromatographic behavior and modified nucleoside content to that of tRNA^Ile purified from cells of the same strain grown in the presence of cysteine. Sulfur-deficient tRNA^Ile (from cysteine-starved cells) was found to have a 5-fold increased V_max in aminoacylation compared to the normal isoacceptor. However, rates or extents of transfer of isoleucine from the [isoleucyl ∼ AMP · Ile-tRNA synthetase] complex were identical with these two tRNAs. Nitrocellulose binding studies suggested that the sulfur-deficient tRNA^Ile bound more efficiently to its synthetase compared to normal tRNA^Ile. Modified nucleoside analysis showed that these tRNAs contained identical amounts of all modified bases except for dihydrouridine and 4-thiouridine. Normal tRNA^Ile contains 1 mol 4-thiouridine and dihydrouridine per mol tRNA, while cysteine-starved tRNA^Ile contains 2 mol dihydrouridine per mol tRNA and is devoid of 4-thiouridine. Several lines of evidence are presented which show that 4-thiouridine can be removed or lost from normal tRNA^Ile without a change in aminoacylation properties. Further, tRNA isolated from E. coli C6 grown with glutathione instead of cysteine has a normal content of 4-thiouridine, but its tRNA^Ile has an increased rate of aminoacylation. We conclude that the low content of dihydrouridine in tRNA^Ile from E. coli cells grown in cysteine-containing medium is most likely responsible for the slow aminoacylation kinetics observed with this tRNA. The possibility that specific dihydrouridine residues in this tRNA might be necessary in establishing the correct conformation of tRNA^Ile for aminoacylation is discussed.     

The tRNA A76 Hydroxyl Groups Control Partitioning of the tRNA-dependent Pre- and Post-transfer Editing Pathways in Class I tRNA Synthetase

Aminoacyl-tRNA synthetases catalyze ATP-dependent covalent coupling of cognate amino acids and tRNAs for ribosomal protein synthesis. Escherichia coli isoleucyl-tRNA synthetase (IleRS) exploits both the tRNA-dependent pre- and post-transfer editing pathways to minimize errors in translation. However, the molecular mechanisms by which tRNA^Ile organizes the synthetic site to enhance pre-transfer editing, an idiosyncratic feature of IleRS, remains elusive. Here we show that tRNA^Ile affects both the synthetic and editing reactions localized within the IleRS synthetic site. In a complex with cognate tRNA, IleRS exhibits a 10-fold faster aminoacyl-AMP hydrolysis and a 10-fold drop in amino acid affinity relative to the free enzyme. Remarkably, the specificity against non-cognate valine was not improved by the presence of tRNA in either of these processes. Instead, amino acid specificity is determined by the protein component per se, whereas the tRNA promotes catalytic performance of the synthetic site, bringing about less error-prone and kinetically optimized isoleucyl-tRNA^Ile synthesis under cellular conditions. Finally, the extent to which tRNA^Ile modulates activation and pre-transfer editing is independent of the intactness of its 3′-end. This finding decouples aminoacylation and pre-transfer editing within the IleRS synthetic site and further demonstrates that the A76 hydroxyl groups participate in post-transfer editing only. The data are consistent with a model whereby the 3′-end of the tRNA remains free to sample different positions within the IleRS·tRNA complex, whereas the fine-tuning of the synthetic site is attained via conformational rearrangement of the enzyme through the interactions with the remaining parts of the tRNA body.     

Allosteric regulation of tRNA import: interactions between tRNA domains at the inner membrane of Leishmania mitochondria

Import of nucleus-encoded tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania involves recognition of specific import signals by the membrane-bound import machinery. Multiple signals on different tRNA domains may be present, and further, importable RNAs interact positively (Type I) or negatively (Type II) with one another at the inner membrane in vitro. By co-transfection assays, it is shown here that tRNA^Tyr (Type I) transiently stimulates the rate of entry of tRNA^Ile (Type II) into Leishmania mitochondria in transfected cells, and conversely, is inhibited by tRNA^Ile. Truncation and mutagenesis experiments led to the co-localization of the effector and import activities of tRNA^Tyr to the D domain, and those of tRNA^Ile to the variable region–T domain (V-T region), indicating that both activities originate from a single RNA–receptor interaction. A third tRNA, human tRNA^Lys, is imported into Leishmania mitochondria in vitro as well as in vivo. This tRNA has Type I and Type II motifs in the D domain and the V-T region, respectively, and shows both Type I and Type II effector activities. Such dual-type tRNAs may interact simultaneously with the Type I and Type II binding sites of the inner membrane import machinery.     

Sequence organization of the chloroplast ribosomal spacer ofSpinacia oleracea including the 3′ end of the 16S rRNA and the 5′ end of the 23S rRNA

The 2201-bp spacer between the chloroplast ribosomal 16S and 23S genes ofSpinacia oleracea was sequenced. It contains the genes of the tRNA^Ile (GAU) and tRNA^Ala (UGC) which are both interrupted by introns of respectively 728 and 816 bp. These introns belong to the class II according to the classfication of Michel and Dujon [17]. Comparison of the rDNA spacer sequence of maize, tobacco and spinach indicates that no conserved polypeptide is encoded within the introns of the two tRNA genes and that the two main insertions/deletions between the three plants are located within two loops of the class II introns secondary structure, which is therefore conserved. Based on the sequence complementarity observed between the upstream and downstream parts, of the 16S and 23S rRNA genes, RNase III-like secondary structures involved in the processing of the rRNA precursor are proposed.     

Sequences of three transfer RNAs from mosquito mitochondria

The sequences of three transfer RNAs from mosquito cell mitochondria, tRNA_UCG^Arg, tRNA_GUC^Asp, and tRNA_GAU^Ile, determined using a combination of rapid ladder and fingerprinting procedures are reported. These were compared with hamster mitochondrial tRNA_UCG^Arg and tRNA_GUC^Asp determined similarly, and a bovine mitochondrial tRNA_GAU^Ile determined using a somewhat different approach. The primary sequences of the mosquito tRNAs were 35 to 65% homologous to the corresponding mammalian mitochondrial species, and bore little homology to “conventional” (bacterial or eucaryotic cytoplasmic) tRNA. The modification status of the mosquito mitochondrial tRNAs resembled that of mammalian mitochondrial tRNA. The results contribute to the generalization that metazoan mitochondrial tRNA constitutes a distinctive, albeit loosely structured, phylogenetic group.     

牛肝TrnaIle的序列分析和二级结构

用随机降解法和Donis-Keller酶分析法,测定了牛肝tRNA^Ile序列.牛肝tRNA^Ile长77个碱基;G5·G69不配对为其显著特征.依据tRNA螺旋区和环区自由能大小及Holley模型,确定了tRNA^Ile的二级结构.     

Commonality and diversity in tRNA substrate recognition in t6A biogenesis by eukaryotic KEOPSs

N ⁶-Threonylcarbamoyladenosine (t⁶A) is a universal and pivotal tRNA modification. KEOPS in eukaryotes participates in its biogenesis, whose mutations are connected with Galloway-Mowat syndrome. However, the tRNA substrate selection mechanism by KEOPS and t⁶A modification function in mammalian cells remain unclear. Here, we confirmed that all ANN-decoding human cytoplasmic tRNAs harbor a t⁶A moiety. Using t⁶A modification systems from various eukaryotes, we proposed the possible coevolution of position 33 of initiator tRNA^Met and modification enzymes. The role of the universal CCA end in t⁶A biogenesis varied among species. However, all KEOPSs critically depended on C32 and two base pairs in the D-stem. Knockdown of the catalytic subunit OSGEP in HEK293T cells had no effect on the steady-state abundance of cytoplasmic tRNAs but selectively inhibited tRNA^Ile aminoacylation. Combined with in vitro aminoacylation assays, we revealed that t⁶A functions as a tRNA^Ile isoacceptor-specific positive determinant for human cytoplasmic isoleucyl-tRNA synthetase (IARS1). t⁶A deficiency had divergent effects on decoding efficiency at ANN codons and promoted +1 frameshifting. Altogether, our results shed light on the tRNA recognition mechanism, revealing both commonality and diversity in substrate recognition by eukaryotic KEOPSs, and elucidated the critical role of t⁶A in tRNA^Ile aminoacylation and codon decoding in human cells.     

Plant mitochondria use two pathways for the biogenesis of tRNAHis

All tRNA^His possess an essential extra G_–1 guanosine residue at their 5′ end. In eukaryotes after standard processing by RNase P, G_–1 is added by a tRNA^His guanylyl transferase. In prokaryotes, G_–1 is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G_–1 we find here that both maturation pathways can be used. Indeed, tRNA^His with or without a G_–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNA^His precursors at both positions G₊₁ and G_–1. The G_–1 is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNA^His without G_–1 has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNA^His with a G_–1 are recovered. This shows that a previously unreported tRNA^His guanylyltransferase activity is present in plant mitochondria.