Mitochondrial histidyl-tRNA Synthetase Research Articles

Plant mitochondria use two pathways for the biogenesis of tRNA His.

All tRNAHis 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 tRNAHis 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, tRNAHis with or without a G–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNAHis 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 tRNAHis 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 tRNAHis with a G–1 are recovered. This shows that a previously unreported tRNAHis guanylyltransferase activity is present in plant mitochondria.

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

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

A recurrent missense variant in HARS2 results in variable sensorineural hearing loss in three unrelated families

HARS2 encodes mitochondrial histidyl-tRNA synthetase (HARS2), which links histidine to its cognate tRNA in the mitochondrial matrix. Biallelic variants in HARS2 are associated with Perrault syndrome, a rare recessive condition characterized by sensorineural hearing loss in both sexes and primary ovarian insufficiency in 46,XX females. Some individuals with Perrault syndrome have a broader phenotypic spectrum with neurological features, including ataxia and peripheral neuropathy. Here, we report a recurrent variant in HARS2 in association with sensorineural hearing loss. In affected individuals from three unrelated families, the variant HARS2 c.1439G>A p.(Arg480His) is present as a heterozygous variant in trans to a putative pathogenic variant. The low prevalence of the allele HARS2 c.1439G>A p.(Arg480His) in the general population and its presence in three families with hearing loss, confirm the pathogenicity of this variant and illustrate the presentation of Perrault syndrome as nonsyndromic hearing loss in males and prepubertal females.

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.

Biochemical analysis of human tRNAHis guanylyltransferase in mitochondrial tRNAHis maturation

Mitochondria contain their own protein synthesis machinery, which includes mitochondrial tRNA maturation. It has been suggested that mammalian mitochondrial tRNAHis (mtRNAHis) is matured by post-transcriptional addition of guanosine at the −1 position (G−1), which serves as an identity element for mitochondrial histidyl-tRNA synthetase. However, the exact maturation process of mammalian mtRNAHis remains unclear. In cytoplasmic tRNAHis (ctRNAHis) maturation, tRNAHis guanylyltransferase (Thg1) adds a GTP onto the 5′-terminal of ctRNAHis and then removes the 5′-pyrophosphate to yield the mature 5′-monophospholylated G-1-ctRNAHis (pG-1-ctRNAHis). Although mammalian Thg1 is localized to both the cytoplasm and mitochondria, it remains unclear whether mammalian Thg1 plays a role in mtRNAHis maturation in mitochondria. Here, we demonstrated that human Thg1 (hThg1) catalyzes the G-1 addition reaction for both human ctRNAHis and mtRNAHis through recognition of the anticodon. While hThg1 catalyzed consecutive GTP additions to mtRNAHisin vitro, it did not exhibit any activity toward mature pG-1-mtRNAHis. We further found that hThg1 could add a GMP directly to the 5′-terminal of mtRNAHis in a template-dependent manner, but fungal Thg1 could not. Therefore, we hypothesized that acceleration of the pyrophosphate removal activity before or after the G-1 addition reaction is a key feature of hThg1 for maintaining a normal 5′-terminal of mtRNAHis in human mitochondria. This study provided a new insight into the differences between tRNAHis maturation in the cytoplasm and mitochondria of humans.

Serendipity or prepared mind? Recollections of the KOP translocation (1967) and of one form of Perrault syndrome.

The human X/autosome translocation, designated KOP, was discovered by Dr. Philip D. Pallister in Montana in 1967 in a young man with apparent Klinefelter syndrome. Collaboratively it was possible to elucidate the genetic nature of his unprecedented chromosome rearrangement and its developmental effects in mother and son. In retrospect, these clinical and genetic studies at the height of the somatic cell genetics era (Ruddle, Siniscalco, etc.) presented human genetics with a highly productive opportunity to begin gene mapping of autosomes and the X chromosome. The late Victor McKusick considered the discovery of the KOP translocation, as he determined personally in Montana, one of the major transforming events in human genetics. The Perrault syndrome evaluated in two families in Montana and one in Sicily for familial deafness, primary amenorrhea and neurologic impairment (progressive in some), turned out to be heterogeneous. In the "hands" of Dr. M.-C. King of Seattle four forms of Perrault syndrome have been identified. The autosomal recessive mutation present in the P family studied with Dr. Pallister in Helena, turned out to affect the mitochondrial histidyl tRNA synthetase gene present in prokaryotes, annelids, fungi and mammals, hence, must already have been present in LUCA some 3.8 billion years ago.

Perrault Syndrome Is Caused by Recessive Mutations in CLPP, Encoding a Mitochondrial ATP-Dependent Chambered Protease

Perrault syndrome is a genetically and clinically heterogeneous autosomal-recessive condition characterized by sensorineural hearing loss and ovarian failure. By a combination of linkage analysis, homozygosity mapping, and exome sequencing in three families, we identified mutations in CLPP as the likely cause of this phenotype. In each family, affected individuals were homozygous for a different pathogenic CLPP allele: c.433A>C (p.Thr145Pro), c.440G>C (p.Cys147Ser), or an experimentally demonstrated splice-donor-site mutation, c.270+4A>G. CLPP, a component of a mitochondrial ATP-dependent proteolytic complex, is a highly conserved endopeptidase encoded by CLPP and forms an element of the evolutionarily ancient mitochondrial unfolded-protein response (UPR(mt)) stress signaling pathway. Crystal-structure modeling suggests that both substitutions would alter the structure of the CLPP barrel chamber that captures unfolded proteins and exposes them to proteolysis. Together with the previous identification of mutations in HARS2, encoding mitochondrial histidyl-tRNA synthetase, mutations in CLPP expose dysfunction of mitochondrial protein homeostasis as a cause of Perrault syndrome.

Mutations in mitochondrial histidyl tRNA synthetase (HARS2) is not associated with premature ovarian failure in a cohort of 69 patients

Previous linkage and sequencing analysis revealed the genetic basis of Perrault syndrome (ovarian dysgenesis and sensorineural hearing loss) in a non-consanguineous family with five affected siblings. Compound heterozygosity for two highly conserved amino acids, L200V and V368L were found in the mitochondrial histidyl tRNA synthetase (HARS2) gene (Pierce, 2011). Functional studies suggested that the Perrault syndrome in this family was caused by the reduction of HARS2 activity. We hypothesized that these mutations may be overrepresented in women with sporadic premature ovarian failure (POF).

Perrault Sendromunun genetik patogenezi

Dear Editor, Premature ovarian insufficiency (POI) is defined as the occurrence of hypergonadotropic hypoestrogenic amenorrhoea in women under the age of 40 years and it is part of well described autosomal recessive syndromes (Perrault, Richard Rundle, Alstrom, Wolfram syndromes and Mitochondriopathies) (1). The association of hypergonadotropic hypogonadism (HH) in females and sensorineural hearing loss (SNHL) in females and males was described as Perrault syndrome (PS). Some patients also have neurological manifestations, but their exact frequency cannot be ascertained since several reports did not include a description of a neurological examination. More recent studies have investigated whether the neurological signs in some of the patients are a coincidental finding or part of the syndrome. Some researchers proposed a possible classification of PS to type I, without neurological disease, and type II or AAHH (The association of ataxia, hypergonadotropic hypergonadism and hearing loss), with progressive neurological disease (2, 3). Previously, partial deficiency of the mitochondrial enzyme cytochrome c oxidase and muscle coenzyme Q10 (CoQ10) deficiency was reported in cases with PS (4, 5). Recently, mutations in 17 beta-hydroxysteroid dehydrogenase type 4 (also known as D-bifunctional protein (HSD17B4/DBP)) which is also involved in Zellweger syndrome, one of three leukodystrophies and mitochondrial histidyl tRNA synthetase (HARS2) also implicated, in the leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL), have been proposed as the genetic causes of PS (6–8). The “ovarioleukodystrophies” comprise a group of rare leukodystrophies associated with POI. Some of the patients have a variant of “vanishing white matter disease” with mutations in subunits of eukaryotic initiation factor 2B (EIF2B) (9). Although HSD17B4, mitochondrial histidyl tRNA synthetase (HARS2) and EIF2B should be examined in other cases, it may be time to classify PS type II under the leukodystrophies. The report of Pierce et al. (8), represents a valuable contribution, because recent findings in genetic research have suggested that a large number of genetic disorders are highly related in the genotypical root. For example; Alstrom syndrome has begun to be classsified as a ciliopathy (10). The late-onset form of GAII and the myopathic form of CoQ10 deficiency are allelic diseases and chylomicron retention disease and Marinesco-Sjogren syndrome are related (11, 12). We suggest that, before proceeding with further laboratory investigations, clinical and neurological examinations must be fully performed and a correct diagnosis of the cases made as there are many syndromes sharing several findings. Long term follow up is very important, since some clinical manifestations appear later in life. Pierce et al. (8) evaluated the sisters MK and LK, whose clinical manifestations had been thoroughly described previously by Fiumara et al. (13) and Mc Carthy and Opitz (14). Therefore, these sisters are the only cases who have such a detailed clinical history, multidiciplinary approach and long term follow up. At the end, a causative mutation was discovered (6). This simple approach will provide an opportunity to recognize associated syndromes and evidence requiring the initiation of further laboratory investigations. Before making high cost moleculer analysis; simple blood tests for glucose, vitamin E, folate and B12 levels, alpha-fetoprotein, very long chain fatty acids and phytanic acid, lysosomal enzymes, amino and organic acids, serum ammonia, arterial pH levels, and X-ray of the skeleton could be carried out. Performing ECG, muscle biopsy, MRI, measurement of mitochondrial enzyme cytochrome c oxidase and CoQ10 levels and DNA analysis for trinucleotide expansions at the SCA 1, 2, 3, 6, 7 and FrieDreich’s Ataxia loci, mutations analysis of FMR1, HSD17B4/DBP, mitochondrial HARS2 and EIF2B for a number of similar cases will give valuable information about the pathogenesis of PS (3, 15). PS type II seems to be caused by both a malfunction of the mitochondria and of myelination. The question remains whether there is one gene or at least two different genes responsible for two different clinical entities.

Mutations in mitochondrial histidyl tRNA synthetase HARS2 cause ovarian dysgenesis and sensorineural hearing loss of Perrault syndrome

Perrault syndrome is a genetically heterogeneous recessive disorder characterized by ovarian dysgenesis and sensorineural hearing loss. In a nonconsanguineous family with five affected siblings, linkage analysis and genomic sequencing revealed the genetic basis of Perrault syndrome to be compound heterozygosity for mutations in the mitochondrial histidyl tRNA synthetase HARS2 at two highly conserved amino acids, L200V and V368L. The nucleotide substitution creating HARS2 p.L200V also created an alternate splice leading to deletion of 12 codons from the HARS2 message. Affected family members thus carried three mutant HARS2 transcripts. Aminoacylation activity of HARS2 p.V368L and HARS2 p.L200V was reduced and the deletion mutant was not stably expressed in mammalian mitochondria. In yeast, lethality of deletion of the single essential histydyl tRNA synthetase HTS1 was fully rescued by wild-type HTS1 and by HTS1 p.L198V (orthologous to HARS2 p.L200V), partially rescued by HTS1 p.V381L (orthologous to HARS2 p.V368L), and not rescued by the deletion mutant. In Caenorhabditis elegans, reduced expression by RNAi of the single essential histydyl tRNA synthetase hars-1 severely compromised fertility. Together, these data suggest that Perrault syndrome in this family was caused by reduction of HARS2 activity. These results implicate aberrations of mitochondrial translation in mammalian gonadal dysgenesis. More generally, the relationship between HARS2 and Perrault syndrome illustrates how causality may be demonstrated for extremely rare inherited mutations in essential, highly conserved genes.

Plant mitochondria use two pathways for the biogenesis of tRNA His

All tRNAHis 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 tRNAHis 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, tRNAHis with or without a G–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNAHis 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 tRNAHis 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 tRNAHis with a G–1 are recovered. This shows that a previously unreported tRNAHis guanylyltransferase activity is present in plant mitochondria.

HTS1 encodes both the cytoplasmic and mitochondrial histidyl-tRNA synthetase of Saccharomyces cerevisiae: mutations alter the specificity of compartmentation.

Genetic and biochemical evidence shows that a single nuclear gene HTS1 encodes both the mitochondrial and cytoplasmic histidyl-tRNA synthetases (Hts). The gene specifies two messages, one with two in-frame ATGs (-60 and +1) and another with only the downstream ATG (+1). We have made a new set of mutations that enables us to express only the mitochondrial or the cytoplasmic form and compared the subcellular distribution of the Hts1 protein in these mutants and wild type, using an antibody that interacts with both the mitochondrial and cytoplasmic Hts1 as well as Hts1::LacZ fusions. Mutations in the upstream ATG (-60) or frameshift mutations in the presequence affect only the mitochondrial enzyme and not the cytoplasmic enzyme. Mutations in the downstream ATG (+1 ATG to ATC) destroy the function of the cytosolic enzyme, but do not affect the function of the mitochondrial enzyme. Overexpression of this construct restores cytoplasmic function. Cells expressing a truncated form of Hts containing a deletion of the first 20 amino-terminal residues (Htsc) produce a functional cytoplasmic enzyme, which does not provide mitochondrial function. Overexpression of this truncated cytoplasmic protein provides mitochondrial function and produces detectable levels of the synthetase in the mitochondrion. These experiments suggest that Hts1 contains two domains that together allow efficient localization of Htsm to the mitochondrion: an amino-terminal presequence in the mitochondrial precursor that is likely cleaved upon delivery to the mitochondrion and a second amino-terminal sequence (residues 21-53) present in both the precursor and the cytoplasmic form. Neither one by itself is sufficient to act as an efficient mitochondrial targeting signal. Using our antibody we have been able to detect a protein of increased molecular mass that corresponds to that of the predicted precursor. Taken together these studies show that the specificity of compartmentation of the Hts protein depends upon both the primary sequence and the concentration of the protein in the cell.