An expanded class of histidine-accepting viral tRNA-like structures.

Structured RNA elements are common in the genomes of RNA viruses, often playing critical roles during viral infection. Some viral RNA elements use forms of tRNA mimicry, but the diverse ways this mimicry can be achieved are poorly understood. Histidine-accepting tRNA-like structures (TLSHis) are examples found at the 3' termini of some positive-sense single-stranded RNA (+ssRNA) viruses where they interact with several host proteins, induce histidylation of the RNA genome, and facilitate processes important for infection, to include genome replication. As only five TLSHis examples had been reported, we explored the possible larger phylogenetic distribution and diversity of this TLS class using bioinformatic approaches. We identified many new examples of TLSHis, yielding a rigorous consensus sequence and secondary structure model that we validated by chemical probing of representative TLSHis RNAs. We confirmed new examples as authentic TLSHis by demonstrating their ability to be histidylated in vitro, then used mutational analyses to imply a tertiary interaction that is likely analogous to the D- and T-loop interaction found in canonical tRNAs. These results expand our understanding of how diverse RNA sequences achieve tRNA-like structure and function in the context of viral RNA genomes and lay the groundwork for high-resolution structural studies of tRNA mimicry by histidine-accepting TLSs.

Crystal structure of human cytoplasmic tRNAHis-specific 5'-monomethylphosphate capping enzyme.

BCDIN3 domain containing RNA methyltransferase, BCDIN3D, monomethylates the 5'-monophosphate of cytoplasmic tRNAHis with a G-1:A73 mispair at the top of an eight-nucleotide-long acceptor helix, using S-adenosyl-l-methionine (SAM) as a methyl group donor. In humans, BCDIN3D overexpression is associated with the tumorigenic phenotype and poor prognosis in breast cancer. Here, we present the crystal structure of human BCDIN3D complexed with S-adenosyl-l-homocysteine. BCDIN3D adopts a classical Rossmann-fold methyltransferase structure. A comparison of the structure with that of the closely related methylphosphate capping enzyme, MePCE, which monomethylates the 5'-γ-phosphate of 7SK RNA, revealed the important residues for monomethyl transfer from SAM onto the 5'-monophosphate of tRNAHis and for tRNAHis recognition by BCDIN3D. A structural model of tRNAHis docking onto BCDIN3D suggested the molecular mechanism underlying the different activities between BCDIN3D and MePCE. A loop in BCDIN3D is shorter, as compared to the corresponding region that forms an α-helix to recognize the 5'-end of RNA in MePCE, and the G-1:A73 mispair in tRNAHis allows the N-terminal α-helix of BCDIN3D to wedge the G-1:A73 mispair of tRNAHis. As a result, the 5'-monophosphate of G-1 of tRNAHis is deep in the catalytic pocket for 5'-phosphate methylation. Thus, BCDIN3D is a tRNAHis-specific 5'-monomethylphosphate capping enzyme that discriminates tRNAHis from other tRNA species, and the structural information presented in this study also provides the molecular basis for the development of drugs against breast cancers.

Overexpression of mitochondrial histidyl-tRNA synthetase restores mitochondrial dysfunction caused by a deafness-associated tRNAHis mutation.

The deafness-associated m.12201T>C mutation affects the A5-U68 base-pairing within the acceptor stem of mitochondrial tRNAHis The primary defect in this mutation is an alteration in tRNAHis aminoacylation. Here, we further investigate the molecular mechanism of the deafness-associated tRNAHis 12201T>C mutation and test whether the overexpression of the human mitochondrial histidyl-tRNA synthetase gene (HARS2) in cytoplasmic hybrid (cybrid) cells carrying the m.12201T>C mutation reverses mitochondrial dysfunctions. Using molecular dynamics simulations, we demonstrate that the m.12201T>C mutation perturbs the tRNAHis structure and function, supported by decreased melting temperature, conformational changes, and instability of mutated tRNA. We show that the m.12201T>C mutation-induced alteration of aminoacylation tRNAHis causes mitochondrial translational defects and respiratory deficiency. We found that the transfer of HARS2 into the cybrids carrying the m.12201T>C mutation raises the levels of aminoacylated tRNAHis from 56.3 to 75.0% but does not change the aminoacylation of other tRNAs. Strikingly, HARS2 overexpression increased the steady-state levels of tRNAHis and of noncognate tRNAs, including tRNAAla, tRNAGln, tRNAGlu, tRNALeu(UUR), tRNALys, and tRNAMet, in cells bearing the m.12201T>C mutation. This improved tRNA metabolism elevated the efficiency of mitochondrial translation, activities of oxidative phosphorylation complexes, and respiration capacity. Furthermore, HARS2 overexpression markedly increased mitochondrial ATP levels and membrane potential and reduced production of reactive oxygen species in cells carrying the m.12201T>C mutation. These results indicate that HARS2 overexpression corrects the mitochondrial dysfunction caused by the tRNAHis mutation. These findings provide critical insights into the pathophysiology of mitochondrial disease and represent a step toward improved therapeutic interventions for mitochondrial disorders.

Molecular mechanism of substrate recognition and specificity of tRNAHis guanylyltransferase during nucleotide addition in the 3'-5' direction.

The tRNAHis guanylyltransferase (Thg1) transfers a guanosine triphosphate (GTP) in the 3'-5' direction onto the 5'-terminal of tRNAHis, opposite adenosine at position 73 (A73). The guanosine at the -1 position (G-1) serves as an identity element for histidyl-tRNA synthetase. To investigate the mechanism of recognition for the insertion of GTP opposite A73, first we constructed a two-stranded tRNAHis molecule composed of a primer and a template strand through division at the D-loop. Next, we evaluated the structural requirements of the incoming GTP from the incorporation efficiencies of GTP analogs into the two-piece tRNAHis Nitrogen at position 7 and the 6-keto oxygen of the guanine base were important for G-1 addition; however, interestingly, the 2-amino group was found not to be essential from the highest incorporation efficiency of inosine triphosphate. Furthermore, substitution of the conserved A73 in tRNAHis revealed that the G-1 addition reaction was more efficient onto the template containing the opposite A73 than onto the template with cytidine (C73) or other bases forming canonical Watson-Crick base-pairing. Some interaction might occur between incoming GTP and A73, which plays a role in the prevention of continuous templated 3'-5' polymerization. This study provides important insights into the mechanism of accurate tRNAHis maturation.

Minimal requirements for reverse polymerization and tRNA repair by tRNAHis guanylyltransferase.

tRNAHis guanylyltransferase (Thg1) has unique reverse (3'-5') polymerase activity occurring in all three domains of life. Most eukaryotic Thg1 homologs are essential genes involved in tRNAHis maturation. These enzymes normally catalyze a single 5' guanylation of tRNAHis lacking the essential G-1 identity element required for aminoacylation. Recent studies suggest that archaeal type Thg1, which includes most archaeal and bacterial Thg1 enzymes is phylogenetically distant from eukaryotic Thg1. Thg1 is evolutionarily related to canonical 5'-3' forward polymerases but catalyzes reverse 3'-5'polymerization. Similar to its forward polymerase counterparts, Thg1 encodes the conserved catalytic palm domain and fingers domain. Here we investigate the minimal requirements for reverse polymerization. We show that the naturally occurring minimal Thg1 enzyme from Ignicoccus hospitalis (IhThg1), which lacks parts of the conserved fingers domain, is catalytically active. And adds all four natural nucleotides to RNA substrates, we further show that the entire fingers domain of Methanosarcina acetivorans Thg1 and Pyrobaculum aerophilum Thg1 (PaThg1) is dispensable for enzymatic activity. In addition, we identified residues in yeast Thg1 that play a part in preventing extended polymerization. Mutation of these residues with alanine resulted in extended reverse polymerization. PaThg1 was found to catalyze extended, template dependent tRNA repair, adding up to 13 nucleotides to a truncated tRNAHis substrate. Sequencing results suggest that PaThg1 fully restored the near correct sequence of the D- and acceptor stem, but also produced incompletely and incorrectly repaired tRNA products. This research forms the basis for future engineering efforts towards a high fidelity, template dependent reverse polymerase.

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.

Human BCDIN3D monomethylates cytoplasmic histidine transfer RNA.

Human RNA methyltransferase BCDIN3D is overexpressed in breast cancer cells, and is related to the tumorigenic phenotype and poor prognosis of breast cancer. Here, we show that cytoplasmic tRNAHis is the primary target of BCDIN3D in human cells. Recombinant human BCDIN3D, expressed in Escherichia coli, monomethylates the 5΄-monophosphate of cytoplasmic tRNAHis efficiently in vitro. In BCDN3D-knockout cells, established by CRISPR/Cas9 editing, the methyl moiety at the 5΄-monophosphate of cytoplasmic tRNAHis is lost, and the exogenous expression of BCDIN3D in the knockout cells restores the modification in cytoplasmic tRNAHis. BCIDN3D recognizes the 5΄-guanosine nucleoside at position -1 (G-1) and the eight-nucleotide acceptor helix with the G-1-A73 mis-pair at the top of the acceptor stem of cytoplasmic tRNAHis, which are exceptional structural features among cytoplasmic tRNA species. While the monomethylation of the 5΄-monophosphate of cytoplasmic tRNAHis affects neither the overall aminoacylation process in vitro nor the steady-state level of cytoplasmic tRNAHisin vivo, it protects the cytoplasmic tRNAHis transcript from degradation in vitro. Thus, BCDIN3D acts as a cytoplasmic tRNAHis-specific 5΄-methylphosphate capping enzyme. The present results also suggest the possible involvement of the monomethylation of the 5΄-monophosphate of cytoplasmic tRNAHis and/or cytoplasmic tRNAHis itself in the tumorigenesis of breast cancer cells.

5'-Terminal nucleotide variations in human cytoplasmic tRNAHisGUG and its 5'-halves.

Transfer RNAs (tRNAs) are fundamental adapter components of translational machinery. tRNAs can further serve as a source of tRNA-derived noncoding RNAs that play important roles in various biological processes beyond translation. Among all species of tRNAs, tRNAHisGUG has been known to uniquely contain an additional guanosine residue at the -1 position (G-1) of its 5'-end. To analyze this -1 nucleotide in detail, we developed a TaqMan qRT-PCR method that can distinctively quantify human mature cytoplasmic tRNAHisGUG containing G-1, U-1, A-1, or C-1 or lacking the -1 nucleotide (starting from G1). Application of this method to the mature tRNA fraction of BT-474 breast cancer cells revealed the presence of tRNAHisGUG containing U-1 as well as the one containing G-1 Moreover, tRNA lacking the -1 nucleotide was also detected, thus indicating the heterogeneous expression of 5'-tRNAHisGUG variants. A sequence library of sex hormone-induced 5'-tRNA halves (5'-SHOT-RNAs), identified via cP-RNA-seq of a BT-474 small RNA fraction, also demonstrated the expression of 5'-tRNAHisGUG halves containing G-1, U-1, or G1 as 5'-terminal nucleotides. Although the detected 5'-nucleotide species were identical, the relative abundances differed widely between mature tRNA and 5'-half from the same BT-474 cells. The majority of mature tRNAs contained the -1 nucleotide, whereas the majority of 5'-halves lacked this nucleotide, which was biochemically confirmed using a primer extension assay. These results reveal the novel identities of tRNAHisGUG molecules and provide insights into tRNAHisGUG maturation and the regulation of tRNA half production.

Structural basis for recognition of G-1-containing tRNA by histidyl-tRNA synthetase.

Aminoacyl-tRNA synthetases (aaRSs) play a crucial role in protein translation by linking tRNAs with cognate amino acids. Among all the tRNAs, only tRNA(His) bears a guanine base at position -1 (G-1), and it serves as a major recognition element for histidyl-tRNA synthetase (HisRS). Despite strong interests in the histidylation mechanism, the tRNA recognition and aminoacylation details are not fully understood. We herein present the 2.55 Å crystal structure of HisRS complexed with tRNA(His), which reveals that G-1 recognition is principally nonspecific interactions on this base and is made possible by an enlarged binding pocket consisting of conserved glycines. The anticodon triplet makes additional specific contacts with the enzyme but the rest of the loop is flexible. Based on the crystallographic and biochemical studies, we inferred that the uniqueness of histidylation system originates from the enlarged binding pocket (for the extra base G-1) on HisRS absent in other aaRSs, and this structural complementarity between the 5' extremity of tRNA and enzyme is probably a result of coevolution of both.

A deafness-associated tRNAHis mutation alters the mitochondrial function, ROS production and membrane potential.

In this report, we investigated the molecular genetic mechanism underlying the deafness-associated mitochondrial tRNAHis 12201T>C mutation. The destabilization of a highly conserved base-pairing (5A-68U) by the m.12201T>C mutation alters structure and function of tRNAHis. Using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mtDNA-less (ρo) cells, we showed ∼70% decrease in the steady-state level of tRNAHis in mutant cybrids, compared with control cybrids. The mutation changed the conformation of tRNAHis, as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. However, ∼60% increase in aminoacylated level of tRNAHis was observed in mutant cells. The failure in tRNAHis metabolism was responsible for the variable reductions in seven mtDNA-encoded polypeptides in mutant cells, ranging from 37 to 81%, with the average of ∼46% reduction, as compared with those of control cells. The impaired mitochondrial translation caused defects in respiratory capacity in mutant cells. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cells. The data provide the evidence for a mitochondrial tRNAHis mutation leading to deafness.

Structural studies of a bacterial tRNA(HIS) guanylyltransferase (Thg1)-like protein, with nucleotide in the activation and nucleotidyl transfer sites.

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

The amyloid-ß-SDR5C1(ABAD) interaction does not mediate a specific inhibition of mitochondrial RNase P.

The amyloid-ß peptide (Aß) is suggested to cause mitochondrial dysfunction in Alzheimer's disease. The mitochondrial dehydrogenase SDR5C1 (also known as ABAD) was shown to bind Aß and was proposed to thereby mediate mitochondrial toxicity, but the molecular mechanism has not been clarified. We recently identified SDR5C1 as an essential component of human mitochondrial RNase P and its associated tRNA:m¹R9 methyltransferase, the enzymes responsible for tRNA 5'-end processing and methylation of purines at tRNA position 9, respectively. With this work we investigated whether SDR5C1's role as a subunit of these two tRNA-maturation activities represents the mechanistic link between Aß and mitochondrial dysfunction. Using recombinant enzyme components, we tested RNase P and methyltransferase activity upon titration of Aß. Micromolar concentrations of monomeric or oligomerized Aß were required to inhibit tRNA 5'-end processing and position 9 methylation catalyzed by the SDR5C1-containing enzymes, yet similar concentrations of Aß also inhibited related RNase P and methyltransferase activities, which do not contain an SDR5C1 homolog. In conclusion, the proposed deleterious effect of Aß on mitochondrial function cannot be explained by a specific inhibition of mitochondrial RNase P or its tRNA:m¹R9 methyltransferase subcomplex, and the molecular mechanism of SDR5C1-mediated Aß toxicity remains unclear.

Absence of a universal element for tRNAHis identity in Acanthamoeba castellanii.

The additional G(-1) nucleotide on tRNA(His) is a nearly universal feature that specifies tRNA(His) identity in all three domains of life. In eukaryotes, the G(-1) identity element is obtained by a post-transcriptional pathway, through the unusual 3'-5' polymerase activity of the highly conserved tRNA(His) guanylyltransferase (Thg1) enzyme, and no examples of eukaryotic histidyl-tRNAs that lack this essential element have been identified. Here we report that the eukaryote Acanthamoeba castellanii lacks the G(-1) identity element on its tRNA(His), consistent with the lack of a gene encoding a bona fide Thg1 ortholog in the A. castellanii genome. Moreover, the cytosolic histidyl-tRNA synthetase in A. castellanii exhibits an unusual tRNA substrate specificity, efficiently aminoacylating tRNA(His) regardless of the presence of G(-1). A. castellanii does contain two Thg1-related genes (encoding Thg1-like proteins, TLPs), but the biochemical properties we associate here with these proteins are consistent with a function for these TLPs in separate pathways unrelated to tRNA(His) metabolism, such as mitochondrial tRNA repair during 5'-editing.

Mechanism of the activation step of the aminoacylation reaction: a significant difference between class I and class II synthetases.

In the present work we report, for the first time, a novel difference in the molecular mechanism of the activation step of aminoacylation reaction between the class I and class II aminoacyl tRNA synthetases (aaRSs). The observed difference is in the mode of nucleophilic attack by the oxygen atom of the carboxylic group of the substrate amino acid (AA) to the αP atom of adenosine triphosphate (ATP). The syn oxygen atom of the carboxylic group attacks the α-phosphorous atom (αP) of ATP in all class I aaRSs (except TrpRS) investigated, while the anti oxygen atom attacks in the case of class II aaRSs. The class I aaRSs investigated are GluRS, GlnRS, TyrRS, TrpRS, LeuRS, ValRS, IleRS, CysRS, and MetRS and class II aaRSs investigated are HisRS, LysRS, ProRS, AspRS, AsnRS, AlaRS, GlyRS, PheRS, and ThrRS. The variation of the electron density at bond critical points as a function of the conformation of the attacking oxygen atom measured by the dihedral angle ψ (C(α)-C') conclusively proves this. The result shows that the strength of the interaction of syn oxygen and αP is stronger than the interaction with the anti oxygen for class I aaRSs. This indicates that the syn oxygen is the most probable candidate for the nucleophilic attack in class I aaRSs. The result is further supported by the computation of the variation of the nonbonded interaction energies between αP atom and anti oxygen as well as syn oxygen in class I and II aaRSs, respectively. The difference in mechanism is explained based on the analysis of the electrostatic potential of the AA and ATP which shows that the relative arrangement of the ATP with respect to the AA is opposite in class I and class II aaRSs, which is correlated with the organization of the active site in respective aaRSs. A comparative study of the reaction mechanisms of the activation step in a class I aaRS (Glutaminyl tRNA synthetase) and in a class II aaRS (Histidyl tRNA synthetase) is carried out by the transition state analysis. The atoms in molecule analysis of the interaction between active site residues or ions and substrates are carried out in the reactant state and the transition state. The result shows that the observed novel difference in the mechanism is correlated with the organizations of the active sites of the respective aaRSs. The result has implication in understanding the experimentally observed different modes of tRNA binding in the two classes of aaRSs.

tRNAHis-guanylyltransferase establishes tRNAHis identity.

Histidine transfer RNA (tRNA) is unique among tRNA species as it carries an additional nucleotide at its 5' terminus. This unusual G(-1) residue is the major tRNA(His) identity element, and essential for recognition by the cognate histidyl-tRNA synthetase to allow efficient His-tRNA(His) formation. In many organisms G(-1) is added post-transcriptionally as part of the tRNA maturation process. tRNA(His) guanylyltransferase (Thg1) specifically adds the guanylyate residue by recognizing the tRNA(His) anticodon. Thg1 homologs from all three domains of life have been the subject of exciting research that gave rise to a detailed biochemical, structural and phylogenetic enzyme characterization. Thg1 homologs are phylogenetically classified into eukaryal- and archaeal-type enzymes differing characteristically in their cofactor requirements and specificity. Yeast Thg1 displays a unique but limited ability to add 2-3 G or C residues to mutant tRNA substrates, thus catalyzing a 3' → 5' RNA polymerization. Archaeal-type Thg1, which has been horizontally transferred to certain bacteria and few eukarya, displays a more relaxed substrate range and may play additional roles in tRNA editing and repair. The crystal structure of human Thg1 revealed a fascinating structural similarity to 5' → 3' polymerases, indicating that Thg1 derives from classical polymerases and evolved to assume its specific function in tRNA(His) processing.

RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase.

In a cell, peptidyl-tRNA molecules that have prematurely dissociated from ribosomes need to be recycled. This work is achieved by an enzyme called peptidyl-tRNA hydrolase. To characterize the RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase, minimalist substrates inspired from tRNA(His) have been designed and produced. Two minisubstrates consist of an N-blocked histidylated RNA minihelix or a small RNA duplex mimicking the acceptor and TψC stem regions of tRNA(His). Catalytic efficiency of the hydrolase toward these two substrates is reduced by factors of 2 and 6, respectively, if compared with N-acetyl-histidyl-tRNA(His). In contrast, with an N-blocked histidylated microhelix or a tetraloop missing the TψC arm, efficiency of the hydrolase is reduced 20-fold. NMR mapping of complex formation between the hydrolase and the small RNA duplex indicates amino acid residues sensitive to RNA binding in the following: (i) the enzyme active site region; (ii) the helix-loop covering the active site; (iii) the region including Leu-95 and the bordering residues 111-117, supposed to form the boundary between the tRNA core and the peptidyl-CCA moiety-binding sites; (iv) the region including Lys-105 and Arg-133, two residues that are considered able to clamp the 5'-phosphate of tRNA, and (v) the positively charged C-terminal helix (residues 180-193). Functional value of these interactions is assessed taking into account the catalytic properties of various engineered protein variants, including one in which the C-terminal helix was simply subtracted. A strong role of Lys-182 in helix binding to the substrate is indicated.

A protein shuttle system to target RNA into mitochondria.

Mitochondria play a key role in essential cellular functions. A deeper understanding of mitochondrial molecular processes is hampered by the difficulty of incorporating foreign nucleic acids into organelles. Mitochondria of most eukaryotic species import cytosolic tRNAs. Based on this natural process, we describe here a powerful shuttle system to internalize several types of RNAs into isolated mitochondria. We demonstrate that this tool is useful to investigate tRNA processing or mRNA editing in plant mitochondria. Furthermore, we show that the same strategy can be used to address both tRNA and mRNA to isolated mammalian mitochondria. We anticipate our novel approach to be the starting point for various studies on mitochondrial processes. Finally, our study provides new insights into the mechanism of RNA import into mitochondria.

An unusual tRNAThr derived from tRNAHis reassigns in yeast mitochondria the CUN codons to threonine.

The standard genetic code is used by most living organisms, yet deviations have been observed in many genomes, suggesting that the genetic code has been evolving. In certain yeast mitochondria, CUN codons are reassigned from leucine to threonine, which requires an unusual tRNA(Thr) with an enlarged 8-nt anticodon loop ( ). To trace its evolutionary origin we performed a comprehensive phylogenetic analysis which revealed that evolved from yeast mitochondrial tRNA(His). To understand this tRNA identity change, we performed mutational and biochemical experiments. We show that Saccharomyces cerevisiae mitochondrial threonyl-tRNA synthetase (MST1) could attach threonine to both and the regular , but not to the wild-type tRNA(His). A loss of the first nucleotide (G(-1)) in tRNA(His) converts it to a substrate for MST1 with a K(m) value (0.7 µM) comparable to that of (0.3 µM), and addition of G(-1) to allows efficient histidylation by histidyl-tRNA synthetase. We also show that MST1 from Candida albicans, a yeast in which CUN codons remain assigned to leucine, could not threonylate , suggesting that MST1 has coevolved with . Our work provides the first clear example of a recent recoding event caused by alloacceptor tRNA gene recruitment.

tRNA 5'-end repair activities of tRNAHis guanylyltransferase (Thg1)-like proteins from Bacteria and Archaea.

The tRNA(His) guanylyltransferase (Thg1) family comprises a set of unique 3'-5' nucleotide addition enzymes found ubiquitously in Eukaryotes, where they function in the critical G(-1) addition reaction required for tRNA(His) maturation. However, in most Bacteria and Archaea, G(-1) is genomically encoded; thus post-transcriptional addition of G(-1) to tRNA(His) is not necessarily required. The presence of highly conserved Thg1-like proteins (TLPs) in more than 40 bacteria and archaea therefore suggests unappreciated roles for TLP-catalyzed 3'-5' nucleotide addition. Here, we report that TLPs from Bacillus thuringiensis (BtTLP) and Methanosarcina acetivorans (MaTLP) display biochemical properties consistent with a prominent role in tRNA 5'-end repair. Unlike yeast Thg1, BtTLP strongly prefers addition of missing N(+1) nucleotides to 5'-truncated tRNAs over analogous additions to full-length tRNA (k(cat)/K(M) enhanced 5-160-fold). Moreover, unlike for -1 addition, BtTLP-catalyzed additions to truncated tRNAs are not biased toward addition of G, and occur with tRNAs other than tRNA(His). Based on these distinct biochemical properties, we propose that rather than functioning solely in tRNA(His) maturation, bacterial and archaeal TLPs are well-suited to participate in tRNA quality control pathways. These data support more widespread roles for 3'-5' nucleotide addition reactions in biology than previously expected.

Change of tRNA identity leads to a divergent orthogonal histidyl-tRNA synthetase/tRNAHis pair.

Mature tRNA(His) has at its 5'-terminus an extra guanylate, designated as G(-1). This is the major recognition element for histidyl-tRNA synthetase (HisRS) to permit acylation of tRNA(His) with histidine. However, it was reported that tRNA(His) of a subgroup of α-proteobacteria, including Caulobacter crescentus, lacks the critical G(-1) residue. Here we show that recombinant C. crescentus HisRS allowed complete histidylation of a C. crescentus tRNA(His) transcript (lacking G(-1)). The addition of G(-1) did not improve aminoacylation by C. crescentus HisRS. However, mutations in the tRNA(His) anticodon caused a drastic loss of in vitro histidylation, and mutations of bases A73 and U72 also reduced charging. Thus, the major recognition elements in C. crescentus tRNA(His) are the anticodon, the discriminator base and U72, which are recognized by the divergent (based on sequence similarity) C. crescentus HisRS. Transplantation of these recognition elements into an Escherichia coli tRNA(His) template, together with addition of base U20a, created a competent substrate for C. crescentus HisRS. These results illustrate how a conserved tRNA recognition pattern changed during evolution. The data also uncovered a divergent orthogonal HisRS/tRNA(His) pair.