Structural insights into the decoding capability of isoleucine tRNAs with lysidine and agmatidine.

The anticodon modifications of transfer RNAs (tRNAs) finetune the codon recognition on the ribosome for accurate translation. Bacteria and archaea utilize the modified cytidines, lysidine (L) and agmatidine (agm2C), respectively, in the anticodon of tRNAIle to decipher AUA codon. L and agm2C contain long side chains with polar termini, but their functions remain elusive. Here we report the cryogenic electron microscopy structures of tRNAsIle recognizing the AUA codon on the ribosome. Both modifications interact with the third adenine of the codon via a unique C-A geometry. The side chains extend toward 3' direction of the mRNA, and the polar termini form hydrogen bonds with 2'-OH of the residue 3'-adjacent to the AUA codon. Biochemical analyses demonstrated that AUA decoding is facilitated by the additional interaction between the polar termini of the modified cytidines and 2'-OH of the fourth mRNA residue. We also visualized cyclic N6-threonylcarbamoyladenosine (ct6A), another tRNA modification, and revealed a molecular basis how ct6A contributes to efficient decoding.

Mitochondrial haplotype mutation alleviates respiratory defect of MELAS by restoring taurine modification in tRNA with 3243A > G mutation.

The 3243A > G in mtDNA is a representative mutation in mitochondrial diseases. Mitochondrial protein synthesis is impaired due to decoding disorder caused by severe reduction of 5-taurinomethyluridine (τm5U) modification of the mutant mt-tRNALeu(UUR) bearing 3243A > G mutation. The 3243A > G heteroplasmy in peripheral blood reportedly decreases exponentially with age. Here, we found three cases with mild respiratory symptoms despite bearing high rate of 3243A > G mutation (>90%) in blood mtDNA. These patients had the 3290T > C haplotypic mutation in addition to 3243A > G pathogenic mutation in mt-tRNALeu(UUR) gene. We generated cybrid cells of these cases to examine the effects of the 3290T > C mutation on mitochondrial function and found that 3290T > C mutation improved mitochondrial translation, formation of respiratory chain complex, and oxygen consumption rate of pathogenic cells associated with 3243A > G mutation. We measured τm5U frequency of mt-tRNALeu(UUR) with 3243A > G mutation in the cybrids by a primer extension method assisted with chemical derivatization of τm5U, showing that hypomodification of τm5U was significantly restored by the 3290T > C haplotypic mutation. We concluded that the 3290T > C is a haplotypic mutation that suppresses respiratory deficiency of mitochondrial disease by restoring hypomodified τm5U in mt-tRNALeu(UUR) with 3243A > G mutation, implying a potential therapeutic measure for mitochondrial disease associated with pathogenic mutations in mt-tRNAs.

Quality control of protein synthesis in the early elongation stage.

In the early stage of bacterial translation, peptidyl-tRNAs frequently dissociate from the ribosome (pep-tRNA drop-off) and are recycled by peptidyl-tRNA hydrolase. Here, we establish a highly sensitive method for profiling of pep-tRNAs using mass spectrometry, and successfully detect a large number of nascent peptides from pep-tRNAs accumulated in Escherichia coli pthts strain. Based on molecular mass analysis, we found about 20% of the peptides bear single amino-acid substitutions of the N-terminal sequences of E. coli ORFs. Detailed analysis of individual pep-tRNAs and reporter assay revealed that most of the substitutions take place at the C-terminal drop-off site and that the miscoded pep-tRNAs rarely participate in the next round of elongation but dissociate from the ribosome. These findings suggest that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.

Restoration of mitochondrial function through activation of hypomodified tRNAs with pathogenic mutations associated with mitochondrial diseases.

Mutations in mitochondrial (mt-)tRNAs frequently cause mitochondrial dysfunction. Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and myoclonus epilepsy associated with ragged red fibers (MERRF) are major clinical subgroups of mitochondrial diseases caused by pathogenic point mutations in tRNA genes encoded in mtDNA. We previously reported a severe reduction in the frequency of 5-taurinomethyluridine (τm5U) and its 2-thiouridine derivative (τm5s2U) in the anticodons of mutant mt-tRNAs isolated from the cells of patients with MELAS and MERRF, respectively. The hypomodified tRNAs fail to decode cognate codons efficiently, resulting in defective translation of respiratory chain proteins in mitochondria. To restore the mitochondrial activity of MELAS patient cells, we overexpressed MTO1, a τm5U-modifying enzyme, in patient-derived myoblasts. We used a newly developed primer extension method and showed that MTO1 overexpression almost completely restored the τm5U modification of the MELAS mutant mt-tRNALeu(UUR). An increase in mitochondrial protein synthesis and oxygen consumption rate suggested that the mitochondrial function of MELAS patient cells can be activated by restoring the τm5U of the mutant tRNA. In addition, we confirmed that MTO1 expression restored the τm5s2U of the mutant mt-tRNALys in MERRF patient cells. These findings pave the way for epitranscriptomic therapies for mitochondrial diseases.

Intrinsic Ribosome Destabilization Underlies Translation and Provides an Organism with a Strategy of Environmental Sensing.

Nascent polypeptides can modulate the polypeptide elongation speed on the ribosome. Here, we show that nascent chains can even destabilize the translating Escherichia coli ribosome from within. This phenomenon, termed intrinsic ribosome destabilization (IRD), occurs in response to a special amino acid sequence of the nascent chain, without involving the release or the recycling factors. Typically, a consecutive array of acidic residues and those intermitted by alternating prolines induce IRD. The ribosomal protein bL31, which bridges the two subunits, counteracts IRD, such that only strong destabilizing sequences abort translation in living cells. We found that MgtL, the leader peptide of a Mg2+ transporter (MgtA), contains a translation-aborting sequence, which sensitizes the ribosome to a decline in Mg2+ concentration and thereby triggers the MgtA-upregulating genetic scheme. Translation proceeds at an inherent risk of ribosomal destabilization, and nascent chain-ribosome complexes can function as a Mg2+ sensor by harnessing IRD.

Hydroxylation of a conserved tRNA modification establishes non-universal genetic code in echinoderm mitochondria.

The genetic code is not frozen but still evolving, which can result in the acquisition of 'dialectal' codons that deviate from the universal genetic code. RNA modifications in the anticodon region of tRNAs play a critical role in establishing such non-universal genetic codes. In echinoderm mitochondria, the AAA codon specifies asparagine instead of lysine. By analyzing mitochondrial (mt-) tRNALys isolated from the sea urchin (Mesocentrotus nudus), we discovered a novel modified nucleoside, hydroxy-N6-threonylcarbamoyladenosine (ht6A), 3' adjacent to the anticodon (position 37). Biochemical analysis revealed that ht6A37 has the ability to prevent mt-tRNALys from misreading AAA as lysine, thereby indicating that hydroxylation of N6-threonylcarbamoyladenosine (t6A) contributes to the establishment of the non-universal genetic code in echinoderm mitochondria.

A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies.

Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (MRPS23, QRSL1, and PNPLA4) as novel causative genes. We also identified 2 genes known to cause monogenic diseases (MECP2 and TNNI3) and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21) as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder.

LRPPRC/SLIRP suppresses PNPase-mediated mRNA decay and promotes polyadenylation in human mitochondria.

In human mitochondria, 10 mRNAs species are generated from a long polycistronic precursor that is transcribed from the heavy chain of mitochondrial DNA, in theory yielding equal copy numbers of mRNA molecules. However, the steady-state levels of these mRNAs differ substantially. Through absolute quantification of mRNAs in HeLa cells, we show that the copy numbers of all mitochondrial mRNA species range from 6000 to 51,000 molecules per cell, indicating that mitochondria actively regulate mRNA metabolism. In addition, the copy numbers of mitochondrial mRNAs correlated with their cellular half-life. Previously, mRNAs with longer half-lives were shown to be stabilized by the LRPPRC/SLIRP complex, which we find that cotranscriptionally binds to coding sequences of mRNAs. We observed that the LRPPRC/SLIRP complex suppressed 3' exonucleolytic mRNA degradation mediated by PNPase and SUV3. Moreover, LRPPRC promoted the polyadenylation of mRNAs mediated by mitochondrial poly(A) polymerase (MTPAP) in vitro. These findings provide a framework for understanding the molecular mechanism of mRNA metabolism in human mitochondria.

Human mitochondrial tRNAs: biogenesis, function, structural aspects, and diseases.

Mitochondria are eukaryotic organelles that generate most of the energy in the cell by oxidative phosphorylation (OXPHOS). Each mitochondrion contains multiple copies of a closed circular double-stranded DNA genome (mtDNA). Human (mammalian) mtDNA encodes 13 essential subunits of the inner membrane complex responsible for OXPHOS. These mRNAs are translated by the mitochondrial protein synthesis machinery, which uses the 22 species of mitochondrial tRNAs (mt tRNAs) encoded by mtDNA. The unique structural features of mt tRNAs distinguish them from cytoplasmic tRNAs bearing the canonical cloverleaf structure. The genes encoding mt tRNAs are highly susceptible to point mutations, which are a primary cause of mitochondrial dysfunction and are associated with a wide range of pathologies. A large number of nuclear factors involved in the biogenesis and function of mt tRNAs have been identified and characterized, including processing endonucleases, tRNA-modifying enzymes, and aminoacyl-tRNA synthetases. These nuclear factors are also targets of pathogenic mutations linked to various diseases, indicating the functional importance of mt tRNAs for mitochondrial activity.

Human mitochondrial diseases caused by lack of taurine modification in mitochondrial tRNAs.

Mitochondrial DNA mutations that cause mitochondrial dysfunction are responsible for a wide spectrum of human diseases, referred to as mitochondrial diseases. Pathogenic point mutations are found frequently in genes encoding mitochondrial (mt) tRNAs, indicating that impaired functioning of mutant mt tRNAs is the primary cause of mitochondrial dysfunction. Our previous studies revealed the absence of posttranscriptional taurine modification at the anticodon wobble uridine in mutant mt tRNAs isolated from cells derived from patients with two major classes of mitochondrial diseases, MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) and MERRF (myoclonus epilepsy associated with ragged red fibers). Defective taurine modification of the mutant mt tRNAs results in a deficiency in protein synthesis as the cognate codons of the mutant mt tRNA cannot be decoded. These findings represent the first evidence of a molecular pathogenesis caused by an RNA modification disorder.

Crystal structure of a novel JmjC-domain-containing protein, TYW5, involved in tRNA modification.

Wybutosine (yW) is a hypermodified nucleoside found in position 37 of tRNA(Phe), and is essential for correct phenylalanine codon translation. yW derivatives widely exist in eukaryotes and archaea, and their chemical structures have many species-specific variations. Among them, its hydroxylated derivative, hydroxywybutosine (OHyW), is found in eukaryotes including human, but the modification mechanism remains unknown. Recently, we identified a novel Jumonji C (JmjC)-domain-containing protein, TYW5 (tRNA yW-synthesizing enzyme 5), which forms the OHyW nucleoside by carbon hydroxylation, using Fe(II) ion and 2-oxoglutarate (2-OG) as cofactors. In this work, we present the crystal structures of human TYW5 (hTYW5) in the free and complex forms with 2-OG and Ni(II) ion at 2.5 and 2.8 Å resolutions, respectively. The structure revealed that the catalytic domain consists of a ß-jellyroll fold, a hallmark of the JmjC domains and other Fe(II)/2-OG oxygenases. hTYW5 forms a homodimer through C-terminal helix bundle formation, thereby presenting a large, positively charged patch involved in tRNA binding. A comparison with the structures of other JmjC-domain-containing proteins suggested a mechanism for substrate nucleotide recognition. Functional analyses of structure-based mutants revealed the essential Arg residues participating in tRNA recognition by TYW5. These findings extend the repertoire of the tRNA modification enzyme into the Fe(II)/2-OG oxygenase superfamily.

Expanding role of the jumonji C domain as an RNA hydroxylase.

JmjC (Jumonji C) domain-containing proteins are known to be an extensive family of Fe(II)/2-oxoglutarate-dependent oxygenases involved in epigenetic regulation of gene expression by catalyzing oxidative demethylation of methylated histones. We report here that a human JmjC protein named Tyw5p (TYW5) unexpectedly acts in the biosynthesis of a hypermodified nucleoside, hydroxywybutosine, in tRNA(Phe) by catalyzing hydroxylation. The finding provides an insight into the expanding role of JmjC protein as an RNA hydroxylase.

Biogenesis of glutaminyl-mt tRNAGln in human mitochondria.

Mammalian mitochondrial (mt) tRNAs, which are required for mitochondrial protein synthesis, are all encoded in the mitochondrial genome, while mt aminoacyl-tRNA synthetases (aaRSs) are encoded in the nuclear genome. However, no mitochondrial homolog of glutaminyl-tRNA synthetase (GlnRS) has been identified in mammalian genomes, implying that Gln-tRNA(Gln) is synthesized via an indirect pathway in the mammalian mitochondria. We demonstrate here that human mt glutamyl-tRNA synthetase (mtGluRS) efficiently misaminoacylates mt tRNA(Gln) to form Glu-tRNA(Gln). In addition, we have identified a human homolog of the Glu-tRNA(Gln) amidotransferase, the hGatCAB heterotrimer. When any of the hGatCAB subunits were inactivated by siRNA-mediated knock down in human cells, the Glu-charged form of tRNA(Gln) accumulated and defects in respiration could be observed. We successfully reconstituted in vitro Gln-tRNA(Gln) formation catalyzed by the recombinant mtGluRS and hGatCAB. The misaminoacylated form of tRNA(Gln) has a weak binding affinity to the mt elongation factor Tu (mtEF-Tu), indicating that the misaminoacylated form of tRNA(Gln) is rejected from the translational apparatus to maintain the accuracy of mitochondrial protein synthesis.

Measuring mRNA decay in human mitochondria.

Human mitochondria contain a genome encoding 13 proteins, all of which are components of respiratory chain complexes. Mutations in human mitochondrial DNA often have pathological consequences. Although 12 of the mitochondrial mRNAs are generated from the same polycistronic transcript, the steady-state level of each mRNA differs. The stability of each mitochondrial mRNA is post-transcriptionally controlled by polyadenylation and deadenylation. However, the molecular mechanism by which each mRNA attains a unique stability is not fully understood. In this report, we describe a practical method for measuring the half-lives of human mitochondrial mRNAs using quantitative real-time reverse transcription PCR.

Aminoacyl-tRNA surveillance by EF-Tu in mammalian mitochondria.

Aminoacyl-tRNA synthetases specifically recognize their cognate tRNAs and ensure the accuracy of translation. However, in mammalian mitochondria, seryl-tRNA synthetase (mt SerRS) significantly misacylates tRNA(Gln), indicating the presence of another mechanism to be required to maintain the fidelity of mitochondrial protein synthesis. We have revealed that mammalian mitochondrial elongation factor Tu (mt EF-Tu) tends to interact with seryl-tRNA(Gln) with lower affinity than glutaminyl-tRNA(Gln) and seryl-tRNA(Ser). This result proposes that mt EF-Tu has a critical role to maintain the translational fidelity by surveillance of aminoacyl-tRNAs for quality control.