Mitochondrial seryl-tRNA Research Articles

Domain collapse and active site ablation generate a widespread animal mitochondrial seryl-tRNA synthetase.

Through their aminoacylation reactions, aminoacyl tRNA-synthetases (aaRS) establish the rules of the genetic code throughout all of nature. During their long evolution in eukaryotes, additional domains and splice variants were added to what is commonly a homodimeric or monomeric structure. These changes confer orthogonal functions in cellular activities that have recently been uncovered. An unusual exception to the familiar architecture of aaRSs is the heterodimeric metazoan mitochondrial SerRS. In contrast to domain additions or alternative splicing, here we show that heterodimeric metazoan mitochondrial SerRS arose from its homodimeric ancestor not by domain additions, but rather by collapse of an entire domain (in one subunit) and an active site ablation (in the other). The collapse/ablation retains aminoacylation activity while creating a new surface, which is necessary for its orthogonal function. The results highlight a new paradigm for repurposing a member of the ancient tRNA synthetase family.

Mitochondrial RNA m3C methyltransferase METTL8 relies on an isoform-specific N-terminal extension and modifies multiple heterogenous tRNAs

Methyltransferase-like 8 (METTL8) encodes a mitochondria-localized METTL8-Iso1 and a nucleolus-distributed METTL8-Iso4 isoform, which differ only in their N-terminal extension (N-extension), by mRNA alternative splicing. METTL8-Iso1 generates 3-methylcytidine at position 32 (m3C32) of mitochondrial tRNAThr and tRNASer(UCN). Whether METTL8-Iso4 is an active m3C32 methyltransferase and the role of the N-extension in mitochondrial tRNA m3C32 formation remain unclear. Here, we revealed that METTL8-Iso4 was inactive in m3C32 generation due to the lack of N-extension, which contains several absolutely conserved modification-critical residues; the counterparts were likewise essential in cytoplasmic m3C32 biogenesis by methyltransferase-like 2A (METTL2A) or budding yeasts tRNA N3-methylcytidine methyltransferase (Trm140), in vitro and in vivo. Cross-compartment/species tRNA modification assays unexpectedly found that METTL8-Iso1 efficiently introduced m3C32 to several cytoplasmic or even bacterial tRNAs in vitro. m3C32 did not influence tRNAThrN6-threonylcarbamoyladenosine (t6A) modification or aminoacylation. In addition to its interaction with mitochondrial seryl-tRNA synthetase (SARS2), we further discovered an interaction between mitochondrial threonyl-tRNA synthetase (TARS2) and METTL8-Iso1. METTL8-Iso1 substantially stimulated the aminoacylation activities of SARS2 and TARS2 in vitro, suggesting a functional connection between mitochondrial tRNA modification and charging. Altogether, our results deepen the mechanistic insights into mitochondrial m3C32 biogenesis and provide a valuable route to prepare cytoplasmic/bacterial tRNAs with only a m3C32 moiety, aiding in future efforts to investigate its effects on tRNA structure and function.

Genetic Variability of HUPRA Syndrome—A Case Report

HUPRA syndrome is a rare autosomal recessive mitochondrial disorder caused by a mutation in the SARS2 gene encoding mitochondrial seryl-tRNA synthetase (mtSerRS). It includes hyperuricemia, pulmonary hypertension, renal failure, and alkalosis. We present a case report of a boy aged 1 year 2 months with premature anemia, hyperuricemia, pulmonary hypertension, renal failure, and alkalosis and diagnosed with HUPRA syndrome. This disease is known to be progressive and fatal. A genetic test revealed a new previously undescribed heterozygous nucleotide variant in exons 14 and 1 of the SARS2 gene. The nucleotide substitution c.1295G > A (p.Arg432His) was detected in exon 14; according to the criteria of the American College of Medical Genetics (ACMG), this missense mutation is probably pathogenic. The nucleotide substitution c.227T > C (p.Leu76Pro) was detected in exon 1; according to the ACMG criteria, this missense mutation is a variant of unclear significance. We suggest that previously undescribed nucleotide substitutions in the SARS2 gene revealed in a patient with typical clinical presentation of the HUPRA syndrome should be considered as a pathogenic mutation.

Selective degradation of tRNASer(AGY) is the primary driver for mitochondrial seryl-tRNA synthetase-related disease.

Mitochondrial translation is of high significance for cellular energy homeostasis. Aminoacyl-tRNA synthetases (aaRSs) are crucial translational components. Mitochondrial aaRS variants cause various human diseases. However, the pathogenesis of the vast majority of these diseases remains unknown. Here, we identified two novel SARS2 (encoding mitochondrial seryl-tRNA synthetase) variants that cause a multisystem disorder. c.654-14T>A mutation induced mRNA mis-splicing, generating a peptide insertion in the active site; c.1519dupC swapped a critical tRNA-binding motif in the C-terminus due to stop codon readthrough. Both mutants exhibited severely diminished tRNA binding and aminoacylation capacities. A marked reduction in mitochondrial tRNASer(AGY) was observed due to RNA degradation in patient-derived induced pluripotent stem cells (iPSCs), causing impaired translation and comprehensive mitochondrial function deficiencies. These impairments were efficiently rescued by wild-type SARS2 overexpression. Either mutation caused early embryonic fatality in mice. Heterozygous mice displayed reduced muscle tissue-specific levels of tRNASers. Our findings elucidated the biochemical and cellular consequences of impaired translation mediated by SARS2, suggesting that reduced abundance of tRNASer(AGY) is a key determinant for development of SARS2-related diseases.

Structural basis for shape-selective recognition and aminoacylation of a D-armless human mitochondrial tRNA

Human mitochondrial gene expression relies on the specific recognition and aminoacylation of mitochondrial tRNAs (mtRNAs) by nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs). Despite their essential role in cellular energy homeostasis, strong mutation pressure and genetic drift have led to an unparalleled sequence erosion of animal mtRNAs. The structural and functional consequences of this erosion are not understood. Here, we present cryo-EM structures of the human mitochondrial seryl-tRNA synthetase (mSerRS) in complex with mtRNASer(GCU). These structures reveal a unique mechanism of substrate recognition and aminoacylation. The mtRNASer(GCU) is highly degenerated, having lost the entire D-arm, tertiary core, and stable L-shaped fold that define canonical tRNAs. Instead, mtRNASer(GCU) evolved unique structural innovations, including a radically altered T-arm topology that serves as critical identity determinant in an unusual shape-selective readout mechanism by mSerRS. Our results provide a molecular framework to understand the principles of mito-nuclear co-evolution and specialized mechanisms of tRNA recognition in mammalian mitochondrial gene expression.

Molecular basis for human mitochondrial tRNA m3C modification by alternatively spliced METTL8.

METTL8 has recently been identified as the methyltransferase catalyzing 3-methylcytidine biogenesis at position 32 (m3C32) of mitochondrial tRNAs. METTL8 also potentially participates in mRNA methylation and R-loop biogenesis. How METTL8 plays multiple roles in distinct cell compartments and catalyzes mitochondrial tRNA m3C formation remain unclear. Here, we discovered that alternative mRNA splicing generated several isoforms of METTL8. One isoform (METTL8-Iso1) was targeted to mitochondria via an N-terminal pre-sequence, while another one (METTL8-Iso4) mainly localized to the nucleolus. METTL8-Iso1-mediated m3C32 modification of human mitochondrial tRNAThr (hmtRNAThr) was not reliant on t6A modification at A37 (t6A37), while that of hmtRNASer(UCN) critically depended on i6A modification at A37 (i6A37). We clarified the hmtRNAThr substrate recognition mechanism, which was obviously different from that of hmtRNASer(UCN), in terms of requiring a G35 determinant. Moreover, SARS2 (mitochondrial seryl-tRNA synthetase) interacted with METTL8-Iso1 in an RNA-independent manner and modestly accelerated m3C modification activity. We further elucidated how nonsubstrate tRNAs in human mitochondria were efficiently discriminated by METTL8-Iso1. In summary, our results established the expression pattern of METTL8, clarified the molecular basis for m3C32 modification by METTL8-Iso1 and provided the rationale for the involvement of METTL8 in tRNA modification, mRNA methylation or R-loop biogenesis.

Methyltransferase METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs

A subset of eukaryotic tRNAs is methylated in the anticodon loop, forming 3-methylcytosine (m3C) modifications. In mammals, the number of tRNAs containing m3C modifications has been expanded to include mitochondrial (mt) tRNA-Ser-UGA and mt-tRNA-Thr-UGU. However, whereas the enzymes catalyzing m3C formation in nuclear-encoded tRNAs have been identified, the proteins responsible for m3C modification in mt-tRNAs are unknown. Here, we show that m3C formation in human mt-tRNAs is dependent upon the methyltransferase-Like 8 (METTL8) enzyme. We find that METTL8 is a mitochondria-associated protein that interacts with mitochondrial seryl-tRNA synthetase, as well as with mt-tRNAs containing m3C. We demonstrate that human cells deficient in METTL8 exhibit loss of m3C modification in mt-tRNAs, but not nuclear-encoded tRNAs. Consistent with the mitochondrial import of METTL8, the formation of m3C in METTL8-deficient cells could be rescued by re-expression of WT METTL8, but not by a METTL8 variant lacking the N-terminal mitochondrial localization signal. Notably, we found METTL8-deficiency in human cells causes alterations in the native migration pattern of mt-tRNA-Ser-UGA, suggesting a role for m3C in tRNA folding. Altogether, these findings demonstrate that METTL8 is required for m3C formation in mt-tRNAs and uncover a potential function for m3C modification in mitochondrial tRNA structure.

Mitochondrial Protein Synthesis and mtDNA Levels Coordinated through an Aminoacyl-tRNA Synthetase Subunit

The aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARSs) is a central reaction in biology. Multiple regulatory pathways use the aminoacylation status of cytosolic tRNAs to monitor and regulate metabolism. The existence of equivalent regulatory networks within the mitochondria is unknown. Here, we describe a functional network that couples protein synthesis to DNA replication in animal mitochondria. We show that a duplication of the gene coding for mitochondrial seryl-tRNA synthetase (SerRS2) generated in arthropods a paralog protein (SLIMP) that forms a heterodimeric complex with a SerRS2 monomer. This seryl-tRNA synthetase variant is essential for protein synthesis and mitochondrial respiration. In addition, SLIMP interacts with the substrate binding domain of the mitochondrial protease LON, thus stimulating proteolysis of the DNA-binding protein TFAM and preventing mitochondrial DNA (mtDNA) accumulation. Thus, mitochondrial translation is directly coupled to mtDNA levels by a network based upon a profound structural modification of an animal ARS.

Coordination of Mitochondrial Protein Synthesis and DNA Replication Through a Novel Aminoacyl-Trna Synthetase Architecture

The aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARS) is a central reaction in biology. Multiple regulatory pathways utilize the aminoacylation status of cytosolic tRNAs to monitor and regulate metabolism. The existence of equivalent regulatory networks within the mitochondria is unknown. Here we describe a functional network that couples protein synthesis to DNA replication in animal mitochondria. We show that a duplication of the gene coding for mitochondrial seryl-tRNA synthetase (SerRS2) generated a paralog protein (SLIMP) in arthropods that forms a heterodimeric complex with a SerRS2 monomer. This seryl-tRNA synthetase variant is essential for protein synthesis and mitochondrial respiration. In addition, SLIMP interacts with the substrate binding domain of the mitochondrial protease LON, thus stimulating proteolysis of the DNA-binding protein TFAM, and repressing mitochondrial DNA (mtDNA) replication. Thus, mitochondrial translation is directly coupled to mtDNA replication by a network based upon a profound structural modification of an animal ARS.

Natural antisense transcripts are linked to the modulation of mitochondrial function and teliospore dormancy in Ustilago maydis.

The basidiomycete smut fungus Ustilago maydis causes common smut of corn. This disease is spread through the production of teliospores, which are thick-walled dormant structures characterized by low rates of respiration and metabolism. Teliospores are formed when the fungus grows within the plant, and the morphological steps involved in their formation have been described, but the molecular events leading to dormancy are not known. In U. maydis, natural antisense transcripts (NATs) can function to alter gene expression and many NATs have increased levels in the teliospore. One such NAT is as-ssm1 which is complementary to the gene for the mitochondrial seryl-tRNA synthetase (ssm1), an enzyme important to mitochondrial function. The disruption of ssm1 leads to cell lysis, indicating it is also essential for cellular viability. To assess the function of as-ssm1, it was ectopically expressed in haploid cells, where it is not normally present. This expression led to reductions in growth rate, virulence, mitochondrial membrane potential and oxygen consumption. It also resulted in the formation of as-ssm1/ssm1 double-stranded RNA and increased ssm1 transcript levels, but no change in Ssm1 protein levels was detected. Together, these findings suggest a role for as-ssm1 in facilitating teliospore dormancy through dsRNA formation and reduction of mitochondrial function.

Splicing Defect in Mitochondrial Seryl-tRNA Synthetase Gene Causes Progressive Spastic Paresis Instead of HUPRA Syndrome

Mitochondrial aminoacyl-tRNA synthetases are an important group of disease genes typically underlying either a disorder affecting an isolated tissue or a distinct syndrome. Missense mutations in the mitochondrial seryl-tRNA synthetase gene, SARS2, have been identified in HUPRA syndrome (hyperuricemia, pulmonary hypertension, renal failure in infancy, and alkalosis). We report here a homozygous splicing mutation in SARS2 in a patient with progressive spastic paresis. We show that the mutation leads to diminished levels of the synthetase in patient's fibroblasts. This has a destabilizing effect on the tRNASer(AGY) isoacceptor, but to a lesser degree than in HUPRA syndrome patients. tRNASer(UCN) is largely unaffected in both phenotypes. In conclusion, the level of tRNASer(AGY) instability may be a factor in determining tissue manifestation in patients with SARS2 mutations. This finding exemplifies the sensitivity of the nervous system to partially reduced aminoacylation, which is sufficient in other tissues to maintain respiratory chain function.

A new mutation in the gene encoding mitochondrial seryl-tRNA synthetase as a cause of HUPRA syndrome

BackgroundHUPRA syndrome is a rare mitochondrial disease characterized by hyperuricemia, pulmonary hypertension, renal failure in infancy and alkalosis. This syndrome was previously described in three patients with a homozygous mutation c.1169A > G (p.D390G) in SARS2, encoding the mitochondrial seryl-tRNA synthetase.Case presentationHere we report the clinical and genetic findings in a girl and her brother. Both patients were clinically diagnosed with the HUPRA syndrome. Analysis of the pedigree identified a new homozygous mutation c.1205G > A (p.R402H) in SARS2 gene. This mutation is very rare in the population and it is located at the C-terminal globular domain of the homodimeric enzyme very close to p.D390G.ConclusionSeveral data support that p.R402H mutation in SARS2 is a new cause of HUPRA syndrome.

Human mitochondrial disease-like symptoms caused by a reduced tRNA aminoacylation activity in flies

The translation of genes encoded in the mitochondrial genome requires specific machinery that functions in the organelle. Among the many mutations linked to human disease that affect mitochondrial translation, several are localized to nuclear genes coding for mitochondrial aminoacyl-transfer RNA synthetases. The molecular significance of these mutations is poorly understood, but it is expected to be similar to that of the mutations affecting mitochondrial transfer RNAs. To better understand the molecular features of diseases caused by these mutations, and to improve their diagnosis and therapeutics, we have constructed a Drosophila melanogaster model disrupting the mitochondrial seryl-tRNA synthetase by RNA interference. At the molecular level, the knockdown generates a reduction in transfer RNA serylation, which correlates with the severity of the phenotype observed. The silencing compromises viability, longevity, motility and tissue development. At the cellular level, the knockdown alters mitochondrial morphology, biogenesis and function, and induces lactic acidosis and reactive oxygen species accumulation. We report that administration of antioxidant compounds has a palliative effect of some of these phenotypes. In conclusion, the fly model generated in this work reproduces typical characteristics of pathologies caused by mutations in the mitochondrial aminoacylation system, and can be useful to assess therapeutic approaches.

Mutations in the Mitochondrial Seryl-tRNA Synthetase Cause Hyperuricemia, Pulmonary Hypertension, Renal Failure in Infancy and Alkalosis, HUPRA Syndrome

An uncharacterized multisystemic mitochondrial cytopathy was diagnosed in two infants from consanguineous Palestinian kindred living in a single village. The most significant clinical findings were tubulopathy (hyperuricemia, metabolic alkalosis), pulmonary hypertension, and progressive renal failure in infancy (HUPRA syndrome). Analysis of the consanguineous pedigree suggested that the causative mutation is in the nuclear DNA. By using genome-wide SNP homozygosity analysis, we identified a homozygous identity-by-descent region on chromosome 19 and detected the pathogenic mutation c.1169A>G (p.Asp390Gly) in SARS2, encoding the mitochondrial seryl-tRNA synthetase. The same homozygous mutation was later identified in a third infant with HUPRA syndrome. The carrier rate of this mutation among inhabitants of this Palestinian isolate was found to be 1:15. The mature enzyme catalyzes the ligation of serine to two mitochondrial tRNA isoacceptors: tRNA(Ser)(AGY) and tRNA(Ser)(UCN). Analysis of amino acylation of the two target tRNAs, extracted from immortalized peripheral lymphocytes derived from two patients, revealed that the p.Asp390Gly mutation significantly impacts on the acylation of tRNA(Ser)(AGY) but probably not that of tRNA(Ser)(UCN). Marked decrease in the expression of the nonacylated transcript and the complete absence of the acylated tRNA(Ser)(AGY) suggest that this mutation leads to significant loss of function and that the uncharged transcripts undergo degradation.

New Aminoacyl-tRNA Synthetase-like Protein in Insecta with an Essential Mitochondrial Function

Aminoacyl-tRNA synthetases (ARS) are modular enzymes that aminoacylate transfer RNAs (tRNA) for their use by the ribosome during protein synthesis. ARS are essential and universal components of the genetic code that were almost completely established before the appearance of the last common ancestor of all living species. This long evolutionary history explains the growing number of functions being discovered for ARS, and for ARS homologues, beyond their canonical role in gene translation. Here we present a previously uncharacterized paralogue of seryl-tRNA synthetase named SLIMP (seryl-tRNA synthetase-like insect mitochondrial protein). SLIMP is the result of a duplication of a mitochondrial seryl-tRNA synthetase (SRS) gene that took place in early metazoans and was fixed in Insecta. Here we show that SLIMP is localized in the mitochondria, where it carries out an essential function that is unrelated to the aminoacylation of tRNA. The knockdown of SLIMP by RNA interference (RNAi) causes a decrease in respiration capacity and an increase in mitochondrial mass in the form of aberrant mitochondria.

NF-Y influences directionality of transcription from the bidirectional Mrps12/ Sarsm promoter in both mouse and human cells

The bidirectional mammalian promoter for mitoribosomal protein S12 (Mrps12) and mitochondrial seryl-tRNA ligase (Sarsm) contains an array of four CCAAT boxes separated by 34–49 bp. In mouse, these elements were shown previously to interact with transcription factor NF-Y and to be required for efficient transcription. Here we show that the CCAAT boxes of the human promoter also influence relative transcriptional activities in the two directions, although they are not absolutely required for transcription. By mutating CCAAT boxes in all possible permutations, we demonstrate that their function is combinatorial, although not simply additive. EMSA indicated that NF-Y interacts with the array in two alternate ways related to the directional selectivity of transcription. Inversion and/or exchange of individual CCAAT boxes had minimal effects on directional selectivity. Over-expression of wild-type or dominant-negative NF-Y affected transcription in the Sarsm direction only, but in human cells, concomitant expression of dominant-negative constructs for other factors was needed to reveal such effects. We propose that the array of NF-Y type CCAAT boxes maintains bidirectional transcription with an appropriate directional selectivity. Computational analysis confirmed that NF-Y type CCAAT boxes are found preferentially in bidirectional promoters, but many such promoters lack them and must be regulated in another way.

Modulation of Mrps12/ Sarsm promoter activity in response to mitochondrial stress

Transcription from the bidirectional promoter of two mouse genes encoding components of the mitochondrial translational apparatus, mitoribosomal protein S12 (Mrps12) and mitochondrial seryl-tRNA ligase (Sarsm), was shown previously to be dependent upon an array of four CCAAT boxes, interacting with the transcription factor NF-Y. Here we report that the homologous human promoter is governed by a CCAAT box array acting in an essentially similar manner. Analysis of the transcriptional response of both the human and mouse promoters to various mitochondrially acting toxins, including inhibitors of mitochondrial protein synthesis, and agents that bring about uncoupling or respiratory chain inhibition, produced either of two distinct outcomes, depending on the cell type and the conditions used. In mouse C2C12 myoblasts, human HEK293 cells or U2OS osteosarcoma cells, plus HeLa cells at high drug doses or mouse 3T3 fibroblasts subjected to prolonged drug exposure, a dose-dependent, bidirectional suppression of transcription was observed. In 3T3 cells subjected only to pre-treatment with the drugs, bidirectional Mrps12/ Sarsm promoter activity was strongly stimulated. A similar, though weaker stimulation was observed at lower drug doses in HeLa cells. Reporter studies using mutated variants of the mouse promoter construct indicated that the stimulation of promoter activity in response to mitochondrial OXPHOS stress in 3T3 cells was independent of the CCAAT box array and of putative binding sites for NRF-2, AP-1 and other transcription factors, whereas transcriptional downregulation under prolonged mitochondrial stress was CCAAT box-dependent. Promoter stimulation was correlated with mitochondrial ROS production, which may be a crucial component in its signalling.

The bidirectional promoter of two genes for the mitochondrial translational apparatus in mouse is regulated by an array of CCAAT boxes interacting with the transcription factor NF-Y

The genes for mitoribosomal protein S12 (Mrps12) and mitochondrial seryl-tRNA ligase (Sarsm and Sars2) are oppositely transcribed from a conserved promoter region of <200 bp in both human and mouse. Using a dual reporter vector we identified an array of 4 CCAAT box elements required for efficient transcription of the two genes in cultured mouse 3T3 cells, and for enforcing directionality in favour of Mrps12. Electrophoretic mobility shift assay (EMSA) and in vivo footprinting confirmed the importance of these promoter elements as sites of protein-binding, and EMSA supershift and chromatin immunoprecipitation (ChIP) assays identified NF-Y as the key transcription factor involved, revealing a common pattern of protein–DNA interactions in all tissues tested (liver, brain, heart, kidney and 3T3 cells). The inherently bidirectional activity of NF-Y makes it an especially suitable factor to govern promoters of this class, whose expression is linked to cell proliferation.

Crystallization and preliminary X-ray diffraction study of mammalian mitochondrial seryl-tRNA synthetase.

The mitochondrial seryl-tRNA synthetase (mt SerRS) from Bos taurus was overexpressed in Escherichia coli and crystallized using the sitting-drop vapour-diffusion method. Crystals grew in a very narrow range of conditions using PEG 8000 as precipitant at room temperature. An appropriate concentration of lithium sulfate was critical for crystal nucleation. Crystals diffracted well beyond a resolution of 1.6 A and were found to belong to the orthorhombic space group C222(1), with unit-cell parameters a = 79.89, b = 230.42, c = 135.60 A. There is one dimer (M(r) approximately 113 kDa) in the asymmetric unit, with a solvent content of 55%. Efforts to solve the phase problem by molecular replacement are under way.

Genomic organization, expression, and subcellular localization of mouse mitochondrial seryl-tRNA synthetase

We report here the identification and characterization of the mouse mitochondrial seryl-tRNA synthetase (mtSerRS). The genomic organization of mouse mtSerRS has been elucidated. The mouse mtSerRS gene containing 16 exons encodes a 519 residue protein with a strong homology to the mitochondria-like seryl-tRNA synthetase of bacteria, yeast, and other homologs. The mouse mtSerRS is ubiquitously expressed in various tissues, but more abundantly in tissues with high metabolic rates including heart and liver. Surprisingly, this gene, unlike other nuclear genes encoding mitochondrial proteins, exhibited a low expression in skeletal muscle and brain. Furthermore, immunofluorescence analysis of NIH3T3 cells expressing the mtSerRS-GFP fusion protein demonstrated that the mouse mtSerRS localizes in mitochondrion. These observations suggest that the mouse mtSerRS is an evolutionarily conserved protein involved in aminoacylation. Thus, it may play a role in the fidelity in mitochondrial translation and pathogenesis of deafness-associated mutations in the mitochondrial tRNA Ser(UCN).