A Terphenyl Supported Dioxophosphorane Dimer: the Light Congener of Lawesson's and Woollins' Reagents.

Thermolysis of a 1,3-dioxa-2-phospholane supported by the terphenyl ligand AriPr4 (AriPr4 =[C6 H3 -2,6-(C6 H3 -2,6-iPr2 )]) at 150 °C gives [AriPr4 PO2 ]2 via loss of ethene. [AriPr4 PO2 ]2 was characterised by X-ray crystallography and NMR spectroscopy; it contains a 4-membered P-O-P-O ring and is the isostructural oxygen analogue of Lawesson's and Woollins' reagents. The dimeric structure of [AriPr4 PO2 ]2 was found to persist in solution through VT NMR spectroscopy and DOSY, supported by DFT calculations. The addition of DMAP to the 1,3-dioxa-2-phospholane facilitates the loss of ethene to give AriPr4 (DMAP)PO2 after days at room temperature, with this product also characterised by X-ray crystallography and NMR spectroscopy. Replacement of the DMAP with pyridine induces ethene loss from the 1,3-dioxa-2-phospholane to provide gram-scale samples of [AriPr4 PO2 ]2 in 75 % yield in 2 days at only 100 °C.

Defects of mitochondrial RNA turnover lead to the accumulation of double-stranded RNA in vivo.

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology.

Intra-mitochondrial Methylation Deficiency Due to Mutations in SLC25A26.

S-adenosylmethionine (SAM) is the predominant methyl group donor and has a large spectrum of target substrates. As such, it is essential for nearly all biological methylation reactions. SAM is synthesized by methionine adenosyltransferase from methionine and ATP in the cytoplasm and subsequently distributed throughout the different cellular compartments, including mitochondria, where methylation is mostly required for nucleic-acid modifications and respiratory-chain function. We report a syndrome in three families affected by reduced intra-mitochondrial methylation caused by recessive mutations in the gene encoding the only known mitochondrial SAM transporter, SLC25A26. Clinical findings ranged from neonatal mortality resulting from respiratory insufficiency and hydrops to childhood acute episodes of cardiopulmonary failure and slowly progressive muscle weakness. We show that SLC25A26 mutations cause various mitochondrial defects, including those affecting RNA stability, protein modification, mitochondrial translation, and the biosynthesis of CoQ10 and lipoic acid.

SUV3 helicase is required for correct processing of mitochondrial transcripts.

Mitochondrial gene expression is largely regulated by post-transcriptional mechanisms that control the amount and translation of each mitochondrial mRNA. Despite its importance for mitochondrial function, the mechanisms and proteins involved in mRNA turnover are still not fully characterized. Studies in yeast and human cell lines have indicated that the mitochondrial helicase SUV3, together with the polynucleotide phosphorylase, PNPase, composes the mitochondrial degradosome. To further investigate the in vivo function of SUV3 we disrupted the homolog of SUV3 in Drosophila melanogaster (Dm). Loss of dmsuv3 led to the accumulation of mitochondrial mRNAs, without increasing rRNA levels, de novo transcription or decay intermediates. Furthermore, we observed a severe decrease in mitochondrial tRNAs accompanied by an accumulation of unprocessed precursor transcripts. These processing defects lead to reduced mitochondrial translation and a severe respiratory chain complex deficiency, resulting in a pupal lethal phenotype. In summary, our results propose that SUV3 is predominantly required for the processing of mitochondrial polycistronic transcripts in metazoan and that this function is independent of PNPase.

A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome.

Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes.

C7orf30 specifically associates with the large subunit of the mitochondrial ribosome and is involved in translation.

In a comparative genomics study for mitochondrial ribosome-associated proteins, we identified C7orf30, the human homolog of the plant protein iojap. Gene order conservation among bacteria and the observation that iojap orthologs cannot be transferred between bacterial species predict this protein to be associated with the mitochondrial ribosome. Here, we show colocalization of C7orf30 with the large subunit of the mitochondrial ribosome using isokinetic sucrose gradient and 2D Blue Native polyacrylamide gel electrophoresis (BN-PAGE) analysis. We co-purified C7orf30 with proteins of the large subunit, and not with proteins of the small subunit, supporting interaction that is specific to the large mitoribosomal complex. Consistent with this physical association, a mitochondrial translation assay reveals negative effects of C7orf30 siRNA knock-down on mitochondrial gene expression. Based on our data we propose that C7orf30 is involved in ribosomal large subunit function. Sequencing the gene in 35 patients with impaired mitochondrial translation did not reveal disease-causing mutations in C7orf30.

Resolving chaperone-assisted protein folding on the ribosome at the peptide level.

Protein folding in vivo begins during synthesis on the ribosome and is modulated by molecular chaperones that engage the nascent polypeptide. How these features of protein biogenesis influence the maturation pathway of nascent proteins is incompletely understood. Here, we use hydrogen-deuterium exchange mass spectrometry to define, at peptide resolution, the cotranslational chaperone-assisted folding pathway of Escherichia coli dihydrofolate reductase. The nascent polypeptide folds along an unanticipated pathway through structured intermediates not populated during refolding from denaturant. Association with the ribosome allows these intermediates to form, as otherwise destabilizing carboxy-terminal sequences remain confined in the ribosome exit tunnel. Trigger factor binds partially folded states without disrupting their structure, and the nascent chain is poised to complete folding immediately upon emergence of the C terminus from the exit tunnel. By mapping interactions between the nascent chain and ribosomal proteins, we trace the path of the emerging polypeptide during synthesis. Our work reveals new mechanisms by which cellular factors shape the conformational search for the native state.

Termination of protein synthesis in mammalian mitochondria.

All mechanisms of protein synthesis can be considered in four stages: initiation, elongation, termination, and ribosome recycling. Remarkable progress has been made in understanding how these processes are mediated in the cytosol of many species; however, details of organellar protein synthesis remain sketchy. This is an important omission, as defects in human mitochondrial translation are known to cause disease and may contribute to the aging process itself. In this minireview, we focus on the recent advances that have been made in understanding how one of these processes, translation termination, occurs in the human mitochondrion.

Translation termination in human mitochondrial ribosomes.

Mitochondria are ubiquitous and essential organelles for all nucleated cells of higher eukaryotes. They contain their own genome [mtDNA (mitochondrial DNA)], and this autosomally replicating extranuclear DNA encodes a complement of genes whose products are required to couple oxidative phosphorylation. Sequencing of this human mtDNA more than 20 years ago revealed unusual features that included a modified codon usage. Specific deviations from the standard genetic code include recoding of the conventional UGA stop to tryptophan, and, strikingly, the apparent recoding of two arginine triplets (AGA and AGG) to termination signals. This latter reassignment was made because of the absence of cognate mtDNA-encoded tRNAs, and a lack of tRNAs imported from the cytosol. Each of these codons only occurs once and, in both cases, at the very end of an open reading frame. The presence of both AGA and AGG is rarely found in other mammals, and the molecular mechanism that has driven the change from encoding arginine to dictating a translational stop has posed a challenging conundrum. Mitochondria from the majority of other organisms studied use only UAA and UAG, leaving the intriguing question of why human organelles appear to have added the complication of a further two stop codons, AGA and AGG, or have they? In the present review, we report recent data to show that mammalian mitochondria can utilize a -1 frameshift such that only the standard UAA and UAG stop codons are required to terminate the synthesis of all 13 polypeptides.

Adult Onset Leigh Syndrome in the Intensive Care Setting: A Novel Presentation of a C12orf65 Related Mitochondrial Disease.

BACKGROUND: Mitochondrial disease can present at any age, with dysfunction in almost any tissue making diagnosis a challenge. It can result from inherited or sporadic mutations in either the mitochondrial or the nuclear genome, many of which affect intraorganellar gene expression. The estimated prevalence of 1/4300 indicates these to be amongst the commonest inherited neuromuscular disorders, emphasising the importance of recognition of the diagnostic clinical features. OBJECTIVE: Despite major advances in our understanding of the molecular basis of mitochondrial diseases, accurate and early diagnoses are critically dependent on the fastidious clinical and biochemical characterisation of patients. Here we describe a patient harbouring a previously reported homozygous mutation in C12orf65, a mitochondrial protein of unknown function, which does not adhere to the proposed distinct genotype-phenotype relationship. METHODS: We performed clinical, biochemical and molecular analysis including whole exome sequencing on patient samples and cell lines. RESULTS: We report an extremely rare case of an adult presenting with Leigh-like disease, in intensive care, in the 5th decade of life, harbouring a recessively inherited mutation previously reported in children. A global reduction in intra-mitochondrial protein synthesis was observed despite normal or elevated levels of mt-RNA, leading to an isolated complex IV deficiency. CONCLUSIONS: All the reported C12orf65 mutations have shown an autosomal recessive pattern of inheritance. Mitochondrial disease causing mutations inherited in this manner are usually of early onset and associated with a severe, often fatal clinical phenotype. Presentations in adulthood are usually less severe. This patient's late adulthood presentation is in sharp contrast emphasising the clinical variability that is characteristic of mitochondrial disease and illustrates why making a definitive diagnosis remains a formidable challenge.