RNA modification landscape of the human mitochondrial tRNA(LYs) regulates protein synthesis

Post-transcriptional RNA modifications play a critical role in the pathogenesis of human mitochondrial disorders, but the mechanisms by which specific modifications affect mitochondrial protein synthesis remain poorly understood. Here we used a quantitative RNA sequencing approach to investigate, at nucleotide resolution, the stoichiometry and methyl modifications of the entire mitochondrial tRNA pool, and establish the relevance to human disease. We discovered that a N-1 -methyladenosine (m(1)A) modification is missing at position 58 in the mitochondrial tRNA(LYs) of patients with the mitochondrial DNA mutation m.8344 A > G associated with MERRF (myoclonus epilepsy, ragged-red fibers). By restoring the modification on the mitochondrial tRNA(LYs), we demonstrated the importance of the m(1)A58 to translation elongation and the stability of selected nascent chains. Our data indicates regulation of post-transcriptional modifications on mitochondrial tRNAs is finely tuned for the control of mitochondrial gene expression. Collectively, our findings provide novel insight into the regulation of mitochondrial tRNAs and reveal greater complexity to the molecular pathogenesis of MERRF.Peer reviewe

Respiratory chain complex III deficiency due to mutated BCS1L : a novel phenotype with encephalomyopathy, partially phenocopied in a Bcs1l mutant mouse model

Background: Mitochondrial diseases due to defective respiratory chain complex III (CIII) are relatively uncommon. The assembly of the eleven-subunit CIII is completed by the insertion of the Rieske iron-sulfur protein, a process for which BCS1L protein is indispensable. Mutations in the BCS1L gene constitute the most common diagnosed cause of CIII deficiency, and the phenotypic spectrum arising from mutations in this gene is wide. Results: A case of CIII deficiency was investigated in depth to assess respiratory chain function and assembly, and brain, skeletal muscle and liver histology. Exome sequencing was performed to search for the causative mutation(s). The patient's platelets and muscle mitochondria showed respiration defects and defective assembly of CIII was detected in fibroblast mitochondria. The patient was compound heterozygous for two novel mutations in BCS1L, c.306A > T and c.399delA. In the cerebral cortex a specific pattern of astrogliosis and widespread loss of microglia was observed. Further analysis showed loss of Kupffer cells in the liver. These changes were not found in infants suffering from GRACILE syndrome, the most severe BCS1L-related disorder causing early postnatal mortality, but were partially corroborated in a knock-in mouse model of BCS1L deficiency. Conclusions: We describe two novel compound heterozygous mutations in BCS1L causing CIII deficiency. The pathogenicity of one of the mutations was unexpected and points to the importance of combining next generation sequencing with a biochemical approach when investigating these patients. We further show novel manifestations in brain, skeletal muscle and liver, including abnormality in specialized resident macrophages (microglia and Kupffer cells). These novel phenotypes forward our understanding of CIII deficiencies caused by BCS1L mutations.Peer reviewe

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

C6orf203 is an RNA-binding protein involved in mitochondrial protein synthesis.

In all biological systems, RNAs are associated with RNA-binding proteins (RBPs), forming complexes that control gene regulatory mechanisms, from RNA synthesis to decay. In mammalian mitochondria, post-transcriptional regulation of gene expression is conducted by mitochondrial RBPs (mt-RBPs) at various stages of mt-RNA metabolism, including polycistronic transcript production, its processing into individual transcripts, mt-RNA modifications, stability, translation and degradation. To date, only a handful of mt-RBPs have been characterized. Here, we describe a putative human mitochondrial protein, C6orf203, that contains an S4-like domain-an evolutionarily conserved RNA-binding domain previously identified in proteins involved in translation. Our data show C6orf203 to bind highly structured RNA in vitro and associate with the mitoribosomal large subunit in HEK293T cells. Knockout of C6orf203 leads to a decrease in mitochondrial translation and consequent OXPHOS deficiency, without affecting mitochondrial RNA levels. Although mitoribosome stability is not affected in C6orf203-depleted cells, mitoribosome profiling analysis revealed a global disruption of the association of mt-mRNAs with the mitoribosome, suggesting that C6orf203 may be required for the proper maturation and functioning of the mitoribosome. We therefore propose C6orf203 to be a novel RNA-binding protein involved in mitochondrial translation, expanding the repertoire of factors engaged in this process

Absence of the Autophagy Adaptor SQSTM1/p62 Causes Childhood-Onset Neurodegeneration with Ataxia, Dystonia, and Gaze Palsy

Peer reviewe

The Bicoid Stability Factor Controls Polyadenylation and Expression of Specific Mitochondrial mRNAs in Drosophila melanogaster

The bicoid stability factor (BSF) of Drosophila melanogaster has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased de novo transcription. Surprisingly, mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation

ANGEL2 phosphatase activity is required for non-canonical mitochondrial RNA processing.

Canonical RNA processing in mammalian mitochondria is defined by tRNAs acting as recognition sites for nucleases to release flanking transcripts. The relevant factors, their structures, and mechanism are well described, but not all mitochondrial transcripts are punctuated by tRNAs, and their mode of processing has remained unsolved. Using Drosophila and mouse models, we demonstrate that non-canonical processing results in the formation of 3\u27 phosphates, and that phosphatase activity by the carbon catabolite repressor 4 domain-containing family member ANGEL2 is required for their hydrolysis. Furthermore, our data suggest that members of the FAST kinase domain-containing protein family are responsible for these 3\u27 phosphates. Our results therefore propose a mechanism for non-canonical RNA processing in metazoan mitochondria, by identifying the role of ANGEL2

Myoglobinopathy is an adult-onset autosomal dominant myopathy with characteristic sarcoplasmic inclusions

Myoglobin, encoded by MB, is a small cytoplasmic globular hemoprotein highly expressed in cardiac myocytes and oxidative skeletal myofibers. Myoglobin binds O-2, facilitates its intracellular transport and serves as a controller of nitric oxide and reactive oxygen species. Here, we identify a recurrent c.292C>T ( p.His98Tyr) substitution in MB in fourteen members of six European families suffering from an autosomal dominant progressive myopathy with highly characteristic sarcoplasmic inclusions in skeletal and cardiac muscle. Myoglobinopathy manifests in adulthood with proximal and axial weakness that progresses to involve distal muscles and causes respiratory and cardiac failure. Biochemical characterization reveals that the mutant myoglobin has altered O-2 binding, exhibits a faster heme dissociation rate and has a lower reduction potential compared to wild-type myoglobin. Preliminary studies show that mutant myoglobin may result in elevated superoxide levels at the cellular level. These data define a recognizable muscle disease associated with MB mutation.Peer reviewe

Mitochondrial dysfunction in ageing and degenerative disease

The cytoplasm of eukaryotic cells contains a dynamic network of double-membraned organelles, called mitochondria, which perform the process of oxidative phosphorylation (OXPHOS) that provides cellular energy in the form of ATP. The respiratory chain creates an electrochemical gradient across the inner mitochondrial membrane, which drives ATP synthesis by the ATP synthase. Mitochondria are indispensable for normal cell function and survival, and dysfunction of the OXPHOS system can lead to a variety of disease syndromes, collectively termed mitochondrial encephalomyopathies. Mitochondrial dysfunction has also been proposed to be involved in age-associated diseases such as diabetes mellitus, heart disease and neurodegeneration, as well as in the ageing process itself. Tissues with high metabolism seem to be particularly vulnerable to mitochondrial dysfunction and myopathy is one of the common phenotypes in mitochondrial disorders. However, the pathophysiological mechanisms linking respiratory chain deficiency to the various phenotypic manifestations are poorly understood. We therefore generated a mouse model for mitochondrial myopathy by tissue-specific disruption of the nuclear gene encoding mitochondrial transcription factor A (TFAM). These myopathy mice develop a progressive respiratory chain dysfunction in skeletal muscle with typical morphological changes consistent with mitochondrial myopathy. Surprisingly the overall mitochondrial ATP production rate was close to normal in the knockout muscles, likely due to the compensatory increase of mitochondrial mass in the affected muscles. Thus, other factors besides ATP deficiency are likely of importance in mitochondrial myopathy. There is a large number of correlative studies suggesting that mitochondrial dysfunction in skeletal muscle is causing the peripheral insulin resistance observed in patients with diabetes mellitus type 2 (DM2). Unexpectedly, the myopathy mice exhibited normal insulin sensitivity and increased glucose uptake in skeletal muscle, suggesting that reduced respiratory chain function in peripheral tissues may be protective against DM2. The mitochondrial theory of aging proposes that oxidative damage to mitochondrial DNA (mtDNA) leads to mutations and impaired respiratory chain function, which in turn, increases reactive oxygen species (ROS) production. ROS have been suggested to induce oxidative damage to various molecules of the cell and thereby cause the progressive decline seen in ageing. We generated mice expressing a proof-reading-deficient version of the mtDNA polymerase gamma. These mtDNA mutator mice accumulated mtDNA mutations at an increased rate and developed a progressive respiratory chain deficiency. They also developed premature ageing phenotypes and exhibited a reduced lifespan, supporting the suggestion of a causative link between mitochondrial dysfunction and ageing. However, we found no differences in ROS production, no increased expression of ROS scavenging enzymes, and no or minor changes in levels of oxidative damage in cell lines and tissues from the mtDNA mutator mice. We instead propose that the accumulation of mtDNA mutations beyond a critical threshold leads to bioenergetic failure and loss of vital cells. This cell loss caused by respiratory chain dysfunction may lead to reduced organ function and eventually organ failure, giving rise to age-associated disease and important ageing phenotypes