Clinical and molecular characterization of novel FARS2 variants causing neonatal mitochondrial disease.

FARS2 encodes the mitochondrial phenylalanyl-tRNA synthetase (mtPheRS), which is essential for charging mitochondrial (mt-) tRNA with phenylalanine for use in intramitochondrial translation. Many biallelic, pathogenic FARS2 variants have been described previously, which are mostly associated with two distinct clinical phenotypes; an early onset epileptic mitochondrial encephalomyopathy or a later onset spastic paraplegia. In this study, we report on a patient who presented at 3 weeks of age with tachypnoea and poor feeding, which progressed to severe metabolic decompensation with lactic acidosis and seizure activity followed by death at 9 weeks of age.

Four-Color STED Super-Resolution RNA Fluorescent In Situ Hybridization and Immunocytochemistry to Visualize Mitochondrial mRNAs in Context with Mitochondrial Ribosomes.

High-resolution imaging has enabled scientists to explore the mitochondrial network at remarkable resolution. This has been exploited to help increase our knowledge of how mitochondrial gene expression is compartmentalized in cultured cells. Here, we provide detailed methodology to simultaneously visualize up to four components including mtDNA-encoded transcripts, submitochondrial marker proteins, mitoribosomal subunits, or core members of the translational apparatus using STED super-resolution nanoscopy.

Mitochondrial Translation Occurs Preferentially in the Peri-Nuclear Mitochondrial Network of Cultured Human Cells.

Human mitochondria are highly dynamic organelles, fusing and budding to maintain reticular networks throughout many cell types. Although extending to the extremities of the cell, the majority of the network is concentrated around the nucleus in most of the commonly cultured cell lines. This organelle harbours its own genome, mtDNA, with a different gene content to the nucleus, but the expression of which is critical for maintaining oxidative phosphorylation.

High-resolution imaging reveals compartmentalization of mitochondrial protein synthesis in cultured human cells.

Human mitochondria contain their own genome, mitochondrial DNA, that is expressed in the mitochondrial matrix. This genome encodes 13 vital polypeptides that are components of the multisubunit complexes that couple oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane that houses these complexes comprises the inner boundary membrane that runs parallel to the outer membrane, infoldings that form the cristae membranes, and the cristae junctions that separate the two.

Visualizing Mitochondrial Ribosomal RNA and Mitochondrial Protein Synthesis in Human Cell Lines.

Human mitochondria contain their own DNA (mtDNA) that encodes 13 proteins all of which are core subunits of oxidative phosphorylation (OXPHOS) complexes. To form functional complexes, these 13 components need to be correctly assembled with approximately 70 nuclear-encoded subunits that are imported following synthesis in the cytosol. How this complicated coordinated translation and assembly is choreographed is still not clear.

Mitochondrial transplantation-a possible therapeutic for mitochondrial dysfunction?: Mitochondrial transfer is a potential cure for many diseases but proof of efficacy and safety is still lacking.

Transplantation of functional mitochondria directly into defective cells is a novel approach that has recently caught the attention of scientists and the general public alike. Could this be too good to be true?

Messenger RNA delivery to mitoribosomes - hints from a bacterial toxin.

In mammalian mitochondria, messenger RNA is processed and matured from large primary transcripts in structures known as RNA granules. The identity of the factors and process transferring the matured mRNA to the mitoribosome for translation is unclear. Nascent mature transcripts are believed to associate initially with the small mitoribosomal subunit prior to recruitment of the large subunit to form the translationally active monosome.

Breaking a single hydrogen bond in the mitochondrial tRNA -PheRS complex leads to phenotypic pleiotropy of human disease.

Various pathogenic variants in both mitochondrial tRNA and Phenylalanyl-tRNA synthetase mitochondrial protein coding gene (FARS2) gene encoding for the human mitochondrial PheRS have been identified and associated with neurological and/or muscle-related pathologies. An important Guanine-34 (G34)A anticodon mutation associated with myoclonic epilepsy with ragged red fibers (MERRF) syndrome has been reported in hmit-tRNA . The majority of G34 contacts in available aaRSs-tRNAs complexes specifically use that base as an important tRNA identity element.

Rescuing stalled mammalian mitoribosomes - what can we learn from bacteria?

In the canonical process of translation, newly completed proteins escape from the ribosome following cleavage of the ester bond that anchors the polypeptide to the P-site tRNA, after which the ribosome can be recycled to initiate a new round of translation. Not all protein synthesis runs to completion as various factors can impede the progression of ribosomes. Rescuing of stalled ribosomes in mammalian mitochondria, however, does not share the same mechanisms that many bacteria use.

Biallelic Mutations in MTPAP Associated with a Lethal Encephalopathy.

Background: A homozygous founder mutation in /, encoding mitochondrial poly(A) polymerase (MTPAP), was first reported in six individuals of Old Order Amish descent demonstrating an early-onset, progressive spastic ataxia with optic atrophy and learning difficulties. MTPAP contributes to the regulation of mitochondrial gene expression through the polyadenylation of mitochondrially encoded mRNAs. Mitochondrial mRNAs with severely truncated poly(A) tails were observed in affected individuals, and mitochondrial protein expression was altered.

Mammalian mitochondrial translation - revealing consequences of divergent evolution.

Mitochondria are ubiquitous organelles present in the cytoplasm of all nucleated eukaryotic cells. These organelles are described as arising from a common ancestor but a comparison of numerous aspects of mitochondria between different organisms provides remarkable examples of divergent evolution. In humans, these organelles are of dual genetic origin, comprising ∼1500 nuclear-encoded proteins and thirteen that are encoded by the mitochondrial genome.

How much does a disrupted mitochondrial network influence neuronal dysfunction?

Mitochondria are organelles that are present in all nucleated cells in the body. They have manifold functions but famously generate ATP efficiently through the process of oxidative phosphorylation. This ensures all tissues have an adequate energy supply and underlines the need for a fully functional mitochondrial network.

mutations cause mitochondrial encephalopathy and a combined oxidative phosphorylation defect.

OXA1, the mitochondrial member of the YidC/Alb3/Oxa1 membrane protein insertase family, is required for the assembly of oxidative phosphorylation complexes IV and V in yeast. However, depletion of human OXA1 (OXA1L) was previously reported to impair assembly of complexes I and V only. We report a patient presenting with severe encephalopathy, hypotonia and developmental delay who died at 5 years showing complex IV deficiency in skeletal muscle.

Defective mitochondrial protease LonP1 can cause classical mitochondrial disease.

LonP1 is a mitochondrial matrix protease whose selective substrate specificity is essential for maintaining mitochondrial homeostasis. Recessively inherited, pathogenic defects in LonP1 have been previously reported to underlie cerebral, ocular, dental, auricular and skeletal anomalies (CODAS) syndrome, a complex multisystemic and developmental disorder. Intriguingly, although classical mitochondrial disease presentations are well-known to exhibit marked clinical heterogeneity, the skeletal and dental features associated with CODAS syndrome are pathognomonic.

Using mitoribosomal profiling to investigate human mitochondrial translation.

: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNA whilst in humans it is mt-tRNA .

Clinical, biochemical, and genetic features associated with VARS2-related mitochondrial disease.

In recent years, an increasing number of mitochondrial disorders have been associated with mutations in mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), which are key enzymes of mitochondrial protein synthesis. Bi-allelic functional variants in VARS2, encoding the mitochondrial valyl tRNA-synthetase, were first reported in a patient with psychomotor delay and epilepsia partialis continua associated with an oxidative phosphorylation (OXPHOS) Complex I defect, before being described in a patient with a neonatal form of encephalocardiomyopathy. Here we provide a detailed genetic, clinical, and biochemical description of 13 patients, from nine unrelated families, harboring VARS2 mutations.

Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies.

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure.

Human mitochondrial ribosomes can switch structural tRNAs - but when and why?

High resolution cryoEM of mammalian mitoribosomes revealed the unexpected presence of mitochondrially encoded tRNA as a structural component of mitochondrial large ribosomal subunit (mt-LSU). Our previously published data identified that only mitochondrial (mt-) tRNA and mt-tRNA can be incorporated into mammalian mt-LSU and within an organism there is no evidence of tissue specific variation. When mt-tRNA is limiting, human mitoribosomes can integrate mt-tRNA instead to generate a translationally competent monosome.

The human RNA-binding protein RBFA promotes the maturation of the mitochondrial ribosome.

Accurate assembly and maturation of human mitochondrial ribosomes is essential for synthesis of the 13 polypeptides encoded by the mitochondrial genome. This process requires the correct integration of 80 proteins, 1 mt (mitochondrial)-tRNA and 2 mt-rRNA species, the latter being post-transcriptionally modified at many sites. Here, we report that human ribosome-binding factor A (RBFA) is a mitochondrial RNA-binding protein that exerts crucial roles in mitoribosome biogenesis.

Kinetic and structural changes in HsmtPheRS, induced by pathogenic mutations in human FARS2.

Mutations in the mitochondrial aminoacyl-tRNA synthetases (mtaaRSs) can cause profound clinical presentations, and have manifested as diseases with very selective tissue specificity. To date most of the mtaaRS mutations could be phenotypically recognized, such that clinicians could identify the affected mtaaRS from the symptoms alone. Among the recently reported pathogenic variants are point mutations in FARS2 gene, encoding the human mitochondrial PheRS.

Store-Operated Ca Entry Controls Induction of Lipolysis and the Transcriptional Reprogramming to Lipid Metabolism.

Ca signals were reported to control lipid homeostasis, but the Ca channels and pathways involved are largely unknown. Store-operated Ca entry (SOCE) is a ubiquitous Ca influx pathway regulated by stromal interaction molecule 1 (STIM1), STIM2, and the Ca channel ORAI1. We show that SOCE-deficient mice accumulate pathological amounts of lipid droplets in the liver, heart, and skeletal muscle.

The Pseudouridine Synthase RPUSD4 Is an Essential Component of Mitochondrial RNA Granules.

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking.