Executioner caspases restrict mitochondrial RNA-driven Type I IFN induction during chemotherapy-induced apoptosis.

During apoptosis, mitochondrial outer membrane permeabilization (MOMP) enables certain mitochondrial matrix macromolecules to escape into the cytosol. However, the fate of mitochondrial RNA (mtRNA) during apoptosis is unknown. Here, we demonstrate that MOMP results in the cytoplasmic release of mtRNA and that executioner caspases-3 and -7 (casp3/7) prevent cytoplasmic mtRNA from triggering inflammatory signaling. In the setting of genetic or pharmacological casp3/7 inhibition, apoptotic insults result in mtRNA activation of the MDA5/MAVS/IRF3 pathway to drive Type I interferon (IFN) signaling. This pathway is sufficient to activate tumor-intrinsic Type I IFN signaling in immunologically cold cancer models that lack an intact cGAS/STING signaling pathway, promote CD8+ T-cell-dependent anti-tumor immunity, and overcome anti-PD1 refractoriness in vivo. Thus, a key function of casp3/7 is to inhibit inflammation caused by the cytoplasmic release of mtRNA, and pharmacological modulation of this pathway increases the immunogenicity of chemotherapy-induced apoptosis.

mtR_find: A Parallel Processing Tool to Identify and Annotate RNAs Derived from the Mitochondrial Genome.

RNAs originating from mitochondrial genomes are abundant in transcriptomic datasets produced by high-throughput sequencing technologies, primarily in short-read outputs. Specific features of mitochondrial small RNAs (mt-sRNAs), such as non-templated additions, presence of length variants, sequence variants, and other modifications, necessitate the need for the development of an appropriate tool for their effective identification and annotation. We have developed mtR_find, a tool to detect and annotate mitochondrial RNAs, including mt-sRNAs and mitochondria-derived long non-coding RNAs (mt-lncRNA). mtR_find uses a novel method to compute the count of RNA sequences from adapter-trimmed reads. When analyzing the published datasets with mtR_find, we identified mt-sRNAs significantly associated with the health conditions, such as hepatocellular carcinoma and obesity, and we discovered novel mt-sRNAs. Furthermore, we identified mt-lncRNAs in early development in mice. These examples show the immediate impact of miR_find in extracting a novel biological information from the existing sequencing datasets. For benchmarking, the tool has been tested on a simulated dataset and the results were concordant. For accurate annotation of mitochondria-derived RNA, particularly mt-sRNA, we developed an appropriate nomenclature. mtR_find encompasses the mt-ncRNA transcriptomes in unpreceded resolution and simplicity, allowing re-analysis of the existing transcriptomic databases and the use of mt-ncRNAs as diagnostic or prognostic markers in the field of medicine.

Macrophage fumarate hydratase restrains mtRNA-mediated interferon production.

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-ß production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.

TEFM variants impair mitochondrial transcription causing childhood-onset neurological disease.

Mutations in the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA biology. The TEFM gene encodes the mitochondrial transcription elongation factor responsible for enhancing the processivity of mitochondrial RNA polymerase, POLRMT. We report for the first time that TEFM variants are associated with mitochondrial respiratory chain deficiency and a wide range of clinical presentations including mitochondrial myopathy with a treatable neuromuscular transmission defect. Mechanistically, we show muscle and primary fibroblasts from the affected individuals have reduced levels of promoter distal mitochondrial RNA transcripts. Finally, tefm knockdown in zebrafish embryos resulted in neuromuscular junction abnormalities and abnormal mitochondrial function, strengthening the genotype-phenotype correlation. Our study highlights that TEFM regulates mitochondrial transcription elongation and its defect results in variable, tissue-specific neurological and neuromuscular symptoms.

mcPGK1-dependent mitochondrial import of PGK1 promotes metabolic reprogramming and self-renewal of liver TICs.

Liver tumour-initiating cells (TICs) contribute to tumour initiation, metastasis, progression and drug resistance. Metabolic reprogramming is a cancer hallmark and plays vital roles in liver tumorigenesis. However, the role of metabolic reprogramming in TICs remains poorly explored. Here, we identify a mitochondria-encoded circular RNA, termed mcPGK1 (mitochondrial circRNA for translocating phosphoglycerate kinase 1), which is highly expressed in liver TICs. mcPGK1 knockdown impairs liver TIC self-renewal, whereas its overexpression drives liver TIC self-renewal. Mechanistically, mcPGK1 regulates metabolic reprogramming by inhibiting mitochondrial oxidative phosphorylation (OXPHOS) and promoting glycolysis. This alters the intracellular levels of α-ketoglutarate and lactate, which are modulators in Wnt/ß-catenin activation and liver TIC self-renewal. In addition, mcPGK1 promotes PGK1 mitochondrial import via TOM40 interactions, reprogramming metabolism from oxidative phosphorylation to glycolysis through PGK1-PDK1-PDH axis. Our work suggests that mitochondria-encoded circRNAs represent an additional regulatory layer controlling mitochondrial function, metabolic reprogramming and liver TIC self-renewal.

Structural and Molecular Basis for Mitochondrial DNA Replication and Transcription in Health and Antiviral Drug Toxicity.

Human mitochondrial DNA (mtDNA) is a 16.9 kbp double-stranded, circular DNA, encoding subunits of the oxidative phosphorylation electron transfer chain and essential RNAs for mitochondrial protein translation. The minimal human mtDNA replisome is composed of the DNA helicase Twinkle, DNA polymerase γ, and mitochondrial single-stranded DNA-binding protein. While the mitochondrial RNA transcription is carried out by mitochondrial RNA polymerase, mitochondrial transcription factors TFAM and TFB2M, and a transcription elongation factor, TEFM, both RNA transcriptions, and DNA replication machineries are intertwined and control mtDNA copy numbers, cellular energy supplies, and cellular metabolism. In this review, we discuss the mechanisms governing these main pathways and the mtDNA diseases that arise from mutations in transcription and replication machineries from a structural point of view. We also address the adverse effect of antiviral drugs mediated by mitochondrial DNA and RNA polymerases as well as possible structural approaches to develop nucleoside reverse transcriptase inhibitor and ribonucleosides analogs with reduced toxicity.

Modopathies Caused by Mutations in Genes Encoding for Mitochondrial RNA Modifying Enzymes: Molecular Mechanisms and Yeast Disease Models.

In eukaryotes, mitochondrial RNAs (mt-tRNAs and mt-rRNAs) are subject to specific nucleotide modifications, which are critical for distinct functions linked to the synthesis of mitochondrial proteins encoded by mitochondrial genes, and thus for oxidative phosphorylation. In recent years, mutations in genes encoding for mt-RNAs modifying enzymes have been identified as being causative of primary mitochondrial diseases, which have been called modopathies. These latter pathologies can be caused by mutations in genes involved in the modification either of tRNAs or of rRNAs, resulting in the absence of/decrease in a specific nucleotide modification and thus on the impairment of the efficiency or the accuracy of the mitochondrial protein synthesis. Most of these mutations are sporadic or private, thus it is fundamental that their pathogenicity is confirmed through the use of a model system. This review will focus on the activity of genes that, when mutated, are associated with modopathies, on the molecular mechanisms through which the enzymes introduce the nucleotide modifications, on the pathological phenotypes associated with mutations in these genes and on the contribution of the yeast Saccharomyces cerevisiae to confirming the pathogenicity of novel mutations and, in some cases, for defining the molecular defects.

RNA granule-clustered mitochondrial aminoacyl-tRNA synthetases form multiple complexes with the potential to fine-tune tRNA aminoacylation.

Mitochondrial RNA metabolism is suggested to occur in identified compartmentalized foci, i.e. mitochondrial RNA granules (MRGs). Mitochondrial aminoacyl-tRNA synthetases (mito aaRSs) catalyze tRNA charging and are key components in mitochondrial gene expression. Mutations of mito aaRSs are associated with various human disorders. However, the suborganelle distribution, interaction network and regulatory mechanism of mito aaRSs remain largely unknown. Here, we found that all mito aaRSs partly colocalize with MRG, and this colocalization is likely facilitated by tRNA-binding capacity. A fraction of human mitochondrial AlaRS (hmtAlaRS) and hmtSerRS formed a direct complex via interaction between catalytic domains in vivo. Aminoacylation activities of both hmtAlaRS and hmtSerRS were fine-tuned upon complex formation in vitro. We further established a full spectrum of interaction networks via immunoprecipitation and mass spectrometry for all mito aaRSs and discovered interactions between hmtSerRS and hmtAsnRS, between hmtSerRS and hmtTyrRS and between hmtThrRS and hmtArgRS. The activity of hmtTyrRS was also influenced by the presence of hmtSerRS. Notably, hmtSerRS utilized the same catalytic domain in mediating several interactions. Altogether, our results systematically analyzed the suborganelle localization and interaction network of mito aaRSs and discovered several mito aaRS-containing complexes, deepening our understanding of the functional and regulatory mechanisms of mito aaRSs.

SP1 and NFY Regulate the Expression of PNPT1, a Gene Encoding a Mitochondrial Protein Involved in Cancer.

The Polyribonucleotide nucleotidyltransferase 1 gene (PNPT1) encodes polynucleotide phosphorylase (PNPase), a 3'-5' exoribonuclease involved in mitochondrial RNA degradation and surveillance and RNA import into the mitochondrion. Here, we have characterized the PNPT1 promoter by in silico analysis, luciferase reporter assays, electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP), siRNA-based mRNA silencing and RT-qPCR. We show that the Specificity protein 1 (SP1) transcription factor and Nuclear transcription factor Y (NFY) bind the PNPT1 promoter, and have a relevant role regulating the promoter activity, PNPT1 expression, and mitochondrial activity. We also found in Kaplan-Meier survival curves that a high expression of either PNPase, SP1 or NFY subunit A (NFYA) is associated with a poor prognosis in liver cancer. In summary, our results show the relevance of SP1 and NFY in PNPT1 expression, and point to SP1/NFY and PNPase as possible targets in anti-cancer therapy.

Mitochondrial tRNAGln 4394C>T Mutation May Contribute to the Clinical Expression of 1555A>G-Induced Deafness.

The mitochondrial 1555A>G mutation plays a critical role in aminoglycoside-induced and non-syndromic hearing loss (AINSHL). Previous studies have suggested that mitochondrial secondary variants may modulate the clinical expression of m.1555A>G-induced deafness, but the molecular mechanism has remained largely undetermined. In this study, we investigated the contribution of a deafness-associated tRNAGln 4394C>T mutation to the clinical expression of the m.1555A>G mutation. Interestingly, a three-generation family with both the m.1555A>G and m.4394C>T mutations exhibited a higher penetrance of hearing loss than another family harboring only the m.1555A>G mutation. At the molecular level, the m.4394C>T mutation resides within a very conserved nucleotide of tRNAGln, which forms a new base-pairing (7T-66A) and may affect tRNA structure and function. Using trans-mitochondrial cybrid cells derived from three subjects with both the m.1555A>G and m.4394C>T mutations, three patients with only the m.1555A>G mutation and three control subjects without these primary mutations, we observed that cells with both the m.1555A>G and m.4394C>T mutations exhibited more severely impaired mitochondrial functions than those with only the m.1555A>G mutation. Furthermore, a marked decrease in mitochondrial RNA transcripts and respiratory chain enzymes was observed in cells harboring both the m.1555A>G and m.4394C>T mutations. Thus, our data suggest that the m.4394C>T mutation may play a synergistic role in the m.1555A>G mutation, enhancing mitochondrial dysfunctions and contributing to a high penetrance of hearing loss in families with both mtDNA pathogenic mutations.

GRP23 plays a core role in E-type editosomes via interacting with MORFs and atypical PPR-DYWs in Arabidopsis mitochondria.

Identifying the PPR-E+-NUWA-DYW2 editosome improves our understanding of the C-to-U RNA editing in plant organelles. However, the mechanism of RNA editing remains to be elucidated. Here, we report that GLUTAMINE-RICH PROTEIN23 (GRP23), a previously identified nuclear transcription regulator, plays an essential role in mitochondrial RNA editing through interacting with MORF (multiple organellar RNA-editing factor) proteins and atypical DYW-type pentatricopeptide repeat (PPR) proteins. GRP23 is targeted to mitochondria, plastids, and nuclei. Analysis of the grp23 mutants rescued by embryo-specific complementation shows decreased editing efficiency at 352 sites in mitochondria and 6 sites in plastids, with a predominant specificity for sites edited by the PPR-E and PPR-DYW proteins. GRP23 interacts with atypical PPR-DYW proteins (MEF8, MEF8S, DYW2, and DYW4) and MORF proteins (MORF1 and MORF8), whereas the four PPR-DYWs interact with the two MORFs. These interactions may increase the stability of the GRP23-MORF-atypical PPR-DYW complex. Furthermore, analysis of mef8N△64aamef8s double mutants shows that MEF8/MEF8S are required for the editing of the PPR-E protein-targeted sites in mitochondria. GRP23 could enhance the interaction between PPR-E and MEF8/MEF8S and form a homodimer or heterodimer with NUWA. Genetic complementation analysis shows that the C-terminal domains of GRP23 and NUWA possess a similar function, probably in the interaction with the MORFs. NUWA also interacts with atypical PPR-DYWs in yeast. Both GRP23 and NUWA interact with the atypical PPR-DYWs, suggesting that the PPR-E proteins recruit MEF8/MEF8S, whereas the PPR-E+ proteins specifically recruit DYW2 as the trans deaminase, and then GRP23, NUWA, and MORFs facilitate and/or stabilize the E or E+-type editosome formation.

Plant mitochondrial RNA editing factors can perform targeted C-to-U editing of nuclear transcripts in human cells.

RNA editing processes are strikingly different in animals and plants. Up to thousands of specific cytidines are converted into uridines in plant chloroplasts and mitochondria whereas up to millions of adenosines are converted into inosines in animal nucleo-cytosolic RNAs. It is unknown whether these two different RNA editing machineries are mutually incompatible. RNA-binding pentatricopeptide repeat (PPR) proteins are the key factors of plant organelle cytidine-to-uridine RNA editing. The complete absence of PPR mediated editing of cytosolic RNAs might be due to a yet unknown barrier that prevents its activity in the cytosol. Here, we transferred two plant mitochondrial PPR-type editing factors into human cell lines to explore whether they could operate in the nucleo-cytosolic environment. PPR56 and PPR65 not only faithfully edited their native, co-transcribed targets but also different sets of off-targets in the human background transcriptome. More than 900 of such off-targets with editing efficiencies up to 91%, largely explained by known PPR-RNA binding properties, were identified for PPR56. Engineering two crucial amino acid positions in its PPR array led to predictable shifts in target recognition. We conclude that plant PPR editing factors can operate in the entirely different genetic environment of the human nucleo-cytosol and can be intentionally re-engineered towards new targets.

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.

The mitochondrial RNA granule modulates manganese-dependent cell toxicity.

Prolonged manganese exposure causes manganism, a neurodegenerative movement disorder. The identity of adaptive and nonadaptive cellular processes targeted by manganese remains mostly unexplored. Here we study mechanisms engaged by manganese in genetic cellular models known to increase susceptibility to manganese exposure, the plasma membrane manganese efflux transporter SLC30A10 and the mitochondrial Parkinson's gene PARK2. We found that SLC30A10 and PARK2 mutations as well as manganese exposure compromised the mitochondrial RNA granule composition and function, resulting in disruption of mitochondrial transcript processing. These RNA granule defects led to impaired assembly and function of the mitochondrial respiratory chain. Notably, cells that survived a cytotoxic manganese challenge had impaired RNA granule function, thus suggesting that this granule phenotype was adaptive. CRISPR gene editing of subunits of the mitochondrial RNA granule, FASTKD2 or DHX30, as well as pharmacological inhibition of mitochondrial transcription-translation, were protective rather than deleterious for survival of cells acutely exposed to manganese. Similarly, adult Drosophila mutants with defects in the mitochondrial RNA granule component scully were safeguarded from manganese-induced mortality. We conclude that impairment of the mitochondrial RNA granule function is a protective mechanism for acute manganese toxicity.

Structural basis for shape-selective recognition and aminoacylation of a D-armless human mitochondrial tRNA.

Human mitochondrial gene expression relies on the specific recognition and aminoacylation of mitochondrial tRNAs (mtRNAs) by nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs). Despite their essential role in cellular energy homeostasis, strong mutation pressure and genetic drift have led to an unparalleled sequence erosion of animal mtRNAs. The structural and functional consequences of this erosion are not understood. Here, we present cryo-EM structures of the human mitochondrial seryl-tRNA synthetase (mSerRS) in complex with mtRNASer(GCU). These structures reveal a unique mechanism of substrate recognition and aminoacylation. The mtRNASer(GCU) is highly degenerated, having lost the entire D-arm, tertiary core, and stable L-shaped fold that define canonical tRNAs. Instead, mtRNASer(GCU) evolved unique structural innovations, including a radically altered T-arm topology that serves as critical identity determinant in an unusual shape-selective readout mechanism by mSerRS. Our results provide a molecular framework to understand the principles of mito-nuclear co-evolution and specialized mechanisms of tRNA recognition in mammalian mitochondrial gene expression.

A novel splice variant of Elp3/Kat9 regulates mitochondrial tRNA modification and function.

Post-translational modifications, such as lysine acetylation, regulate the activity of diverse proteins across many cellular compartments. Protein deacetylation in mitochondria is catalyzed by the enzymatic activity of the NAD+-dependent deacetylase sirtuin 3 (SIRT3), however it remains unclear whether corresponding mitochondrial acetyltransferases exist. We used a bioinformatics approach to search for mitochondrial proteins with an acetyltransferase catalytic domain, and identified a novel splice variant of ELP3 (mt-ELP3) of the elongator complex, which localizes to the mitochondrial matrix in mammalian cells. Unexpectedly, mt-ELP3 does not mediate mitochondrial protein acetylation but instead induces a post-transcriptional modification of mitochondrial-transfer RNAs (mt-tRNAs). Overexpression of mt-ELP3 leads to the protection of mt-tRNAs against the tRNA-specific RNase angiogenin, increases mitochondrial translation, and furthermore increases expression of OXPHOS complexes. This study thus identifies mt-ELP3 as a non-canonical mt-tRNA modifying enzyme.

Evolution of mitochondrial RNA editing in extant gymnosperms.

To unveil the evolution of mitochondrial RNA editing in gymnosperms, we characterized mitochondrial genomes (mitogenomes), plastid genomes, RNA editing sites, and pentatricopeptide repeat (PPR) proteins from 10 key taxa representing four of the five extant gymnosperm clades. The assembled mitogenomes vary in gene content due to massive gene losses in Gnetum and Conifer II clades. Mitochondrial gene expression levels also vary according to protein function, with the most highly expressed genes involved in the respiratory complex. We identified 9132 mitochondrial C-to-U editing sites, as well as 2846 P-class and 8530 PLS-class PPR proteins. Regains of editing sites were demonstrated in Conifer II rps3 transcripts whose corresponding mitogenomic sequences lack introns due to retroprocessing. Our analyses reveal that non-synonymous editing is efficient and results in more codons encoding hydrophobic amino acids. In contrast, synonymous editing, although performed with variable efficiency, can increase the number of U-ending codons that are preferentially utilized in gymnosperm mitochondria. The inferred loss-to-gain ratio of mitochondrial editing sites in gymnosperms is 2.1:1, of which losses of non-synonymous editing are mainly due to genomic C-to-T substitutions. However, such substitutions only explain a small fraction of synonymous editing site losses, indicating distinct evolutionary mechanisms. We show that gymnosperms have experienced multiple lineage-specific duplications in PLS-class PPR proteins. These duplications likely contribute to accumulated RNA editing sites, as a mechanistic correlation between RNA editing and PLS-class PPR proteins is statistically supported.

Maize Shrek1 encodes a WD40 protein that regulates pre-rRNA processing in ribosome biogenesis.

Ribosome biogenesis is a fundamental and highly orchestrated process that involves hundreds of ribosome biogenesis factors. Despite advances that have been made in yeast, the molecular mechanism of ribosome biogenesis remains largely unknown in plants. We uncovered a WD40 protein, Shrunken and Embryo Defective Kernel 1 (SHREK1), and showed that it plays a crucial role in ribosome biogenesis and kernel development in maize (Zea mays). The shrek1 mutant shows an aborted embryo and underdeveloped endosperm and embryo-lethal in maize. SHREK1 localizes mainly to the nucleolus and accumulates to high levels in the seed. Depleting SHREK1 perturbs pre-rRNA processing and causes imbalanced profiles of mature rRNA and ribosome. The expression pattern of ribosomal-related genes is significantly altered in shrek1. Like its yeast (Saccharomyces cerevisiae) ortholog Periodic tryptophan protein 1 (PWP1), SHREK1 physically interacts with ribosomal protein ZmRPL7a, a transient component of the PWP1-subcomplex involved in pre-rRNA processing in yeast. Additionally, SHREK1 may assist in the A3 cleavage of the pre-rRNA in maize by interacting with the nucleolar protein ZmPOP4, a maize homolog of the yeast RNase mitochondrial RNA-processing complex subunit. Overall, our work demonstrates a vital role of SHREK1 in pre-60S ribosome maturation, and reveals that impaired ribosome function accounts for the embryo lethality in shrek1.

Identification of human mitochondrial RNA cleavage sites and candidate RNA processing factors.

BACKGROUND: The human mitochondrial genome is transcribed as long strands of RNA containing multiple genes, which require post-transcriptional cleavage and processing to release functional gene products that play vital roles in cellular energy production. Despite knowledge implicating mitochondrial post-transcriptional processes in pathologies such as cancer, cardiovascular disease and diabetes, very little is known about the way their function varies on a human population level and what drives changes in these processes to ultimately influence disease risk. Here, we develop a method to detect and quantify mitochondrial RNA cleavage events from standard RNA sequencing data and apply this approach to human whole blood data from > 1000 samples across independent cohorts. RESULTS: We detect 54 putative mitochondrial RNA cleavage sites that not only map to known gene boundaries, short RNA ends and RNA modification sites, but also occur at internal gene positions, suggesting novel mitochondrial RNA cleavage junctions. Inferred RNA cleavage rates correlate with mitochondrial-encoded gene expression across individuals, suggesting an impact on downstream processes. Furthermore, by comparing inferred cleavage rates to nuclear genetic variation and gene expression, we implicate multiple genes in modulating mitochondrial RNA cleavage (e.g. MRPP3, TBRG4 and FASTKD5), including a potentially novel role for RPS19 in influencing cleavage rates at a site near to the MTATP6-COX3 junction that we validate using shRNA knock down data. CONCLUSIONS: We identify novel cleavage junctions associated with mitochondrial RNA processing, as well as genes newly implicated in these processes, and detect the potential impact of variation in cleavage rates on downstream phenotypes and disease processes. These results highlight the complexity of the mitochondrial transcriptome and point to novel mechanisms through which nuclear-encoded genes can potentially influence key mitochondrial processes.