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.

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.

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' 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' phosphates. Our results therefore propose a mechanism for non-canonical RNA processing in metazoan mitochondria, by identifying the role of ANGEL2.

Mutations in the mitochondrial tryptophanyl-tRNA synthetase cause growth retardation and progressive leukoencephalopathy.

BACKGROUND: Mutations in mitochondrial aminoacyl tRNA synthetases form a subgroup of mitochondrial disorders often only perturbing brain function by affecting mitochondrial translation. Here we report two siblings with mitochondrial disease, due to compound heterozygous mutations in the mitochondrial tryptophanyl-tRNA synthetase (WARS2) gene, presenting with severe neurological symptoms but normal mitochondrial function in skeletal muscle biopsies and cultured skin fibroblasts. METHODS: Whole exome sequencing on genomic DNA samples from both subjects and their parents identified two compound heterozygous variants c.833T>G (p.Val278Gly) and c.938A>T (p.Lys313Met) in the WARS2 gene as potential disease-causing variants. We generated patient-derived neuroepithelial stem cells and modeled the disease in yeast and Drosophila melanogaster to confirm pathogenicity. RESULTS: Biochemical analysis of patient-derived neuroepithelial stem cells revealed a mild combined complex I and IV defect, while modeling the disease in yeast demonstrated that the reported aminoacylation defect severely affects respiration and viability. Furthermore, silencing of wild type WARS2 in Drosophila melanogaster showed that a partial defect in aminoacylation is enough to cause lethality. CONCLUSIONS: Our results establish the identified WARS2 variants as disease-causing and highlight the benefit of including human neuronal models, when investigating mutations specifically affecting the nervous system.