Strand Of mtDNA Research Articles

Both-strand gene coding in a plastome-like mitogenome of an enoplid nematode.

The phylum Nematoda remains very poorly sampled for mtDNA, with a strong bias toward parasitic, economically important or model species of the Chromadoria lineage. Most chromadorian mitogenomes share a specific order of genes encoded on one mtDNA strand. However, the few sequenced representatives of the Dorylaimia lineage exhibit a variable order of mtDNA genes encoded on both strands. While the ancestral arrangement of nematode mitogenome remains undefined, no evidence has been reported for Enoplia, the phylum's third early divergent major lineage. We describe the first mitogenome of an enoplian nematode, Campydora demonstrans, and contend that the complete 37-gene repertoire and both-strand gene encoding are ancestral states preserved in Enoplia and Dorylaimia versus the derived mitogenome arrangement in some Chromadoria. The C. demonstrans mitogenome is 17,018 bp in size and contains a noncoding perfect inverted repeat with 2013 bp-long arms, subdividing the mitogenome into two coding regions. This mtDNA arrangement is very rare among animals and instead resembles that of chloroplast genomes in land plants. Our report broadens mtDNA taxonomic sampling of the phylum Nematoda and adds support to the applicability of cox1 gene as a phylogenetic marker for establishing nematode relationships within higher taxa.

Mitochondrial replication's role in vertebrate mtDNA strand asymmetry.

Mitogenomes are defined as compact and structurally stable over aeons. This perception results from a vertebrate-centric vision, where few types of mtDNA rearrangements are described. Here, we bring a new light to the involvement of mitochondrial replication in the strand asymmetry of the vertebrate mtDNA. Using several species of deep-sea hatchetfish (Sternoptychidae) displaying distinct mtDNA structural arrangements, we unravel the inversion of the coding direction of protein-coding genes (PCGs). This unexpected change is coupled with a strand asymmetry nucleotide composition reversal and is shown to be directly related to the strand location of the Control Region (CR). An analysis of the fourfold redundant sites of the PCGs (greater than 6000 vertebrates), revealed the rarity of this phenomenon, found in nine fish species (five deep-sea hatchetfish). Curiously, in Antarctic notothenioid fishes (Trematominae), where a single PCG inversion (the only other record in fish) is coupled with the inversion of the CR, the standard asymmetry is disrupted for the remaining PCGs but not yet reversed, suggesting a transitory state. Our results hint that a relaxation of the classic vertebrate mitochondrial structural stasis promotes disruption of the natural balance of asymmetry of the mtDNA. These findings support the long-lasting hypothesis that replication is the main molecular mechanism promoting the strand-specific compositional bias of this unique and indispensable molecule.

Single-Nucleotide Resolution Mapping of N6-Methyladenine in Genomic DNA.

N6-Methyladenine (6mA) is a naturally occurring DNA modification in both prokaryotes and eukaryotes. Herein, we developed a deaminase-mediated sequencing (DM-seq) method for genome-wide mapping of 6mA at single-nucleotide resolution. The method capitalizes on the selective deamination of adenine, but not 6mA, in DNA mediated by an evolved adenine deaminase, ABE8e. By employing this method, we achieved genome-wide mapping of 6mA in Escherichia coli and in mammalian mitochondrial DNA (mtDNA) at single-nucleotide resolution. We found that the 6mA sites are mainly located in the GATC motif in the E. coli genome. We also identified 17 6mA sites in mtDNA of HepG2 cells, where all of the 6mA sites are distributed in the heavy strand of mtDNA. We envision that DM-seq will be a valuable tool for uncovering new functions of 6mA in DNA and for exploring its potential roles in mitochondria-related human diseases.

Mitochondrial stress in advanced fibrosis and cirrhosis associated with chronic hepatitis B, chronic hepatitis C, or nonalcoholic steatohepatitis.

Hepatitis B virus (HBV) infection causes oxidative stress (OS) and alters mitochondria in experimental models. Our goal was to investigate whether HBV might alter liver mitochondria also in humans, and the resulting mitochondrial stress might account for the progression of fibrosis in chronic hepatitis B (CHB). The study included 146 treatment-naïve CHB mono-infected patients. Patients with CHB and advanced fibrosis (AF) or cirrhosis (F3-F4) were compared to patients with no/mild-moderate fibrosis (F0-F2). Patients with CHB were further compared to patients with chronic hepatitis C (CHC; n = 33), nonalcoholic steatohepatatis (NASH; n = 12), and healthy controls ( n = 24). We detected oxidative damage to mitochondrial DNA (mtDNA), including mtDNA strand beaks, and identified multiple mtDNA deletions in patients with F3-F4 as compared to patients with F0-F2. Alterations in mitochondrial function, mitochondrial unfolded protein response, biogenesis, mitophagy, and liver inflammation were observed in patients with AF or cirrhosis associated with CHB, CHC, and NASH. In vitro , significant increases of the mitochondrial formation of superoxide and peroxynitrite as well as mtDNA damage, nitration of the mitochondrial respiratory chain complexes, and impairment of complex I occurred in HepG2 cells replicating HBV or transiently expressing hepatitits B virus X protein. mtDNA damage and complex I impairment were prevented with the superoxide-scavenging Mito-Tempo or with inducible nitric oxide synthase (iNOS)-specific inhibitor 1400 W. Our results emphasized the importance of mitochondrial OS, mtDNA damage, and associated alterations in mitochondrial function and dynamics in AF or cirrhosis in CHB and NASH. Mitochondria might be a target in drug development to stop fibrosis progression.

Mitochondrial DNA integrity and function are critical for endothelium-dependent vasodilation in rats with metabolic syndrome.

Endothelial dysfunction in diabetes is generally attributed to oxidative stress, but this view is challenged by observations showing antioxidants do not eliminate diabetic vasculopathy. As an alternative to oxidative stress-induced dysfunction, we interrogated if impaired mitochondrial function in endothelial cells is central to endothelial dysfunction in the metabolic syndrome. We observed reduced coronary arteriolar vasodilation to the endothelium-dependent dilator, acetylcholine (Ach), in Zucker Obese Fatty rats (ZOF, 34 ± 15% [mean ± standard deviation] 10-3M) compared to Zucker Lean rats (ZLN, 98 ± 11%). This reduction in dilation occurred concomitantly with mitochondrial DNA (mtDNA) strand lesions and reduced mitochondrial complex activities in the endothelium of ZOF versus ZLN. To demonstrate endothelial dysfunction is linked to impaired mitochondrial function, administration of a cell-permeable, mitochondria-directed endonuclease (mt-tat-EndoIII), to repair oxidatively modified DNA in ZOF, restored mitochondrial function and vasodilation to Ach (94 ± 13%). Conversely, administration of a cell-permeable, mitochondria-directed exonuclease (mt-tat-ExoIII) produced mtDNA strand breaks in ZLN, reduced mitochondrial complex activities and vasodilation to Ach in ZLN (42 ± 16%). To demonstrate that mitochondrial function is central to endothelium-dependent vasodilation, we introduced (via electroporation) liver mitochondria (from ZLN) into the endothelium of a mesenteric vessel from ZOF and restored endothelium-dependent dilation to vasoactive intestinal peptide (VIP at 10-5M, 4 ± 3% vasodilation before mitochondrial transfer and 48 ± 36% after transfer). Finally, to demonstrate mitochondrial function is key to endothelium-dependent dilation, we administered oligomycin (mitochondrial ATP synthase inhibitor) and observed a reduction in endothelium-dependent dilation. We conclude that mitochondrial function is critical for endothelium-dependent vasodilation.

Low guanine content and biased nucleotide distribution in vertebrate mtDNA can cause overestimation of non-CpG methylation.

ABSTRACTMitochondrial DNA (mtDNA) methylation in vertebrates has been hotly debated for over 40 years. Most contrasting results have been reported following bisulfite sequencing (BS-seq) analyses. We addressed whether BS-seq experimental and analysis conditions influenced the estimation of the levels of methylation in specific mtDNA sequences. We found false positive non-CpG methylation in the CHH context (fpCHH) using unmethylated Sus scrofa mtDNA BS-seq data. fpCHH methylation was detected on the top/plus strand of mtDNA within low guanine content regions. These top/plus strand sequences of fpCHH regions would become extremely AT-rich sequences after BS-conversion, whilst bottom/minus strand sequences remained almost unchanged. These unique sequences caused BS-seq aligners to falsely assign the origin of each strand in fpCHH regions, resulting in false methylation calls. fpCHH methylation detection was enhanced by short sequence reads, short library inserts, skewed top/bottom read ratios and non-directional read mapping modes. We confirmed no detectable CHH methylation in fpCHH regions by BS-amplicon sequencing. The fpCHH peaks were located in the D-loop, ATP6, ND2, ND4L, ND5 and ND6 regions and identified in our S. scrofa ovary and oocyte data and human BS-seq data sets. We conclude that non-CpG methylation could potentially be overestimated in specific sequence regions by BS-seq analysis.

The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method

The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues.

Mechanism of transcription initiation and primer generation at the mitochondrial replication origin OriL

The intricate process of human mtDNA replication requires the coordinated action of both transcription and replication machineries. Transcription and replication events at the lagging strand of mtDNA prompt the formation of a stem-loop structure (OriL) and the synthesis of a ∼25nt RNA primer by mitochondrial RNA polymerase (mtRNAP). The mechanisms by which mtRNAP recognizes OriL, initiates transcription, and transfers the primer to the replisome are poorly understood. We found that transcription initiation at OriL involves slippage of the nascent transcript. The transcript slippage is essential for initiation complex stability and its ability to translocate the mitochondrial DNA polymerase gamma, PolG, which pre-binds to OriL, downstream of the replication origin thus allowing for the primer synthesis. Our data suggest the primosome assembly at OriL-a complex of mtRNAP and PolG-can efficiently generate the primer, transfer it to the replisome, and protect it from degradation by mitochondrial endonucleases.

Mitochondrial DNA as a Sensitive Biomarker of UV-Induced Cellular Damage in Human Skin.

Mitochondrial DNA (mtDNA) has been demonstrated to be a reliable biomarker of UV-induced genetic damage in both animal and human skin. Properties of the mitochondrial genome which allow for its use as a biomarker of damage include its presence in multiple copies within a cell, its limited repair mechanisms, and its lack of protective histones. To measure UV-induced mtDNA damage (particularly in the form of strand breaks), real-time quantitative PCR (qPCR) is used, based on the observation that PCR amplification efficiency is decreased in the presence of high levels of damage. Here, we describe the measurement of UV-induced mtDNA damage which includes the extraction of cellular DNA, qPCR to determine the relative amount of mtDNA, qPCR to determine UV-induced damage within a long strand of mtDNA, and the verification of the amplification process using gel electrophoresis.

Mitochondrial DNA damage in calf skeletal muscle and walking performance in people with peripheral artery disease

BackgroundPeripheral artery disease (PAD) is associated with mitochondrial dysfunction in calf skeletal muscle and a greater abundance of mitochondrial DNA (mtDNA) heteroplasmy. However, it is unknown whether calf skeletal muscle mtDNA of PAD participants harbors a greater abundance of mitochondrial DNA 4977-bp common deletion (mtDNA4977), strand breaks and oxidative damage (i.e., oxidized purines) compared to non-PAD participants and whether these mtDNA abnormalities are associated with poor walking performance in participants with PAD. MethodsCalf muscle biopsies were obtained from 50 PAD participants (ankle-brachial index (ABI) < 0.95) and 25 non-PAD participants (ABI = 0.99–1.40) matched by age, sex, and race. The abundance of mtDNA copy number, mtDNA4977 deletion, strand breaks, and oxidized purines in selected mtDNA regions coding for electron transport chain (ETC) constituents and the non-coding D-Loop region was determined in calf muscle. All participants completed measurement of 6-min walk and usual and fast-paced 4-m walking velocity test. ResultsParticipants with PAD (mean age = 65.4 years, SD = 6.9; 14 (28%) women, 38 (76%) black) and without PAD (mean age = 65.2 years, SD = 6.7; 7 (28%) women, 16 (64%) black) did not differ in the abundance of calf muscle mtDNA4977 deletion, mtDNA strand breaks, and oxidized purines. Though, a greater abundance of mtDNA strand breaks within ND4/5 genes was significantly associated with poorer 6-min walk distance, lower usual-paced 4-m walking velocity, and lower fast-paced 4-m walking velocity in non-PAD participants. Significant associations were also found in the density of strand break damage (i.e., damage per mtDNA copy) within ND1/2, ND4/5 and COII/ATPase 6/8 region with 6-min walk distance, usual-paced 4-m walking velocity and fast-paced 4-m walking velocity in non-PAD participants. Significant interactions were found between PAD presence vs. absence and density of strand break damage within ND1/2, ND4/5, COII/ATPase 6/8 regions for the associations with 6-min walk distance, usual-paced 4-m walking velocity, fast-paced 4-m walking velocity. Conversely, of the three walking performance measures only the usual-paced 4-m walking velocity showed a significant, although modest, negative association with the abundance of oxidized purines in the D-Loop (P = 0.031) and ND4/5 (P = 0.033) regions in the calf skeletal muscle of people with PAD. ConclusionOverall, these data suggest that the abundance of calf muscle mtDNA strand breaks and mtDNA4977 common deletion are not associated with walking performance in people with PAD and may not be directly involved in the pathophysiology of PAD. Conversely, strand breaks in specific mtDNA regions may contribute to poor walking performance in people without PAD. Further study is needed to confirm whether usual-paced 4-m walking velocity is associated significantly with a greater abundance of oxidized purines in the D-loop, a “mutational hotspot” for oxidative damage, and why this association may differ from the association with 6-min walk distance and fast-paced 4-m walking velocity.

On separation and coding capacity of mtDNA strands

On separation and coding capacity of mtDNA strands

The strand-biased mitochondrial DNA methylome and its regulation by DNMT3A.

How individual genes are regulated from a mitochondrial polycistronic transcript to have variable expression remains an enigma. Here, through bisulfite sequencing and strand-specific mapping, we show mitochondrial genomes in humans and other animals are strongly biased to light (L)-strand non-CpG methylation with conserved peak loci preferentially located at gene–gene boundaries, which was also independently validated by MeDIP and FspEI digestion. Such mtDNA methylation patterns are conserved across different species and developmental stages but display dynamic local or global changes during development and aging. Knockout of DNMT3A alone perturbed mtDNA regional methylation patterns, but not global levels, and altered mitochondrial gene expression, copy number, and oxygen respiration. Overexpression of DNMT3A strongly increased mtDNA methylation and strand bias. Overall, methylation at gene bodies and boundaries was negatively associated with mitochondrial transcript abundance and also polycistronic transcript processing. Furthermore, HPLC-MS confirmed the methylation signals on mitochondria DNA. Together, these data provide high-resolution mtDNA methylation maps that revealed a strand-specific non-CpG methylation, its dynamic regulation, and its impact on the polycistronic mitochondrial transcript processing.

Mitochondrial DNA Mutations are Associated with Ulcerative Colitis Preneoplasia but Tend to be Negatively Selected in Cancer

The role of mitochondrial DNA (mtDNA) mutations in cancer remains controversial. Ulcerative colitis is an inflammatory bowel disease that increases the risk of colorectal cancer and involves mitochondrial dysfunction, making it an ideal model to study the role of mtDNA in tumorigenesis. Our goal was to comprehensively characterize mtDNA mutations in ulcerative colitis tumorigenesis using Duplex Sequencing, an ultra-accurate next-generation sequencing method. We analyzed 46 colon biopsies from non-ulcerative colitis control patients and ulcerative colitis patients with and without cancer, including biopsies at all stages of dysplastic progression. mtDNA was sequenced at a median depth of 1,364x. Mutations were classified by mutant allele frequency: clonal > 0.95, subclonal 0.01-0.95, and very low frequency (VLF) < 0.01. We identified 208 clonal and subclonal mutations and 56,764 VLF mutations. Mutations were randomly distributed across the mitochondrial genome. Clonal and subclonal mutations increased in number and pathogenicity in early dysplasia, but decreased in number and pathogenicity in cancer. Most clonal, subclonal, and VLF mutations were C>T transitions in the heavy strand of mtDNA, which likely arise from DNA replication errors. A subset of VLF mutations were C>A transversions, which are probably due to oxidative damage. VLF transitions and indels were less abundant in the non-D-loop region and decreased with progression. Our results indicate that mtDNA mutations are frequent in ulcerative colitis preneoplasia but negatively selected in cancers. IMPLICATIONS: While mtDNA mutations might contribute to early ulcerative colitis tumorigenesis, they appear to be selected against in cancer, suggesting that functional mitochondria might be required for malignant transformation in ulcerative colitis.

Tiron is Protective Against ROS-Induced Damage in Human Bronchial Epithelial Cells

Background Reactive oxygen species (ROS)-induced damage and resultant oxidative stress have been documented within the lung and have been implicated in the development of pulmonary diseases such as chronic obstructive pulmonary disorder and asthma (1). Antioxidant supplementation has been used to inhibit accumulation of oxidative damage such as DNA strand breaks and lipid peroxidation in inflammation (1,2). Tiron, a superoxide scavenger, has previously been shown to reduce murine airway remodelling and associated inflammation as well as offer protection against UV and H2O2-induced mitochondrial DNA (mtDNA) damage in human dermal fibroblasts (2,3). Aim This study aimed to investigate the use of the Tiron to prevent ROS-induced damage in human bronchial epithelial cells (BEAS-2B). Methods BEAS-2B cells were pre-treated with 3mM Tiron in BEGM (Lonza, UK) for 24 hours prior to induction of ROS damage (1 hour H2O2 (0.25mM) or exposure to 120 hour hypoxic (1% O2) conditions). Simultaneous Tiron treatment and ROS-induction was also assessed. The level of ROS production was assessed via flow cytometric analysis using the DCFDA assay (Abcam, UK). MtDNA strand breaks were assessed using a qPCR 1kb amplification following 83bp alignment (4). Concentration of the lipid peroxidation biomarker malondialdehyde (MDA) was quantified using a colourimetric assay (Sigma, UK). All data was normalised to the cell controls where appropriate and summarised as mean ±SEM, analysed by one-way ANOVA and Dunnett’s posthoc test (p≤0.05). Results Both pre-treatment and the addition of Tiron at the time of ROS-induction demonstrated 100% protection (±0.18 vs. H2O2; ±0.06 vs. hypoxia pre-treatment and ±0.30 co-treatment) against both H2O2 and hypoxia induced mtDNA strand breaks. This was supported by a significant (p≤0.001) reduction in the production of ROS and diminished formation of MDA with both H2O2 and hypoxic conditions compared to untreated controls. Conclusion This study demonstrates a promising use for Tiron as a supplement against both H2O2 and hypoxia-induced ROS in BEAS-2B cells. However, the precise mechanism of Tiron’s action requires further elucidation. The findings of this study indicate a potential use for Tiron in the treatment and prevention of pulmonary diseases associated with oxidative stress.

Increased TFAM binding to mtDNA damage hot spots is associated with mtDNA loss in aged rat heart

The well-known age-related mitochondrial dysfunction deeply affects heart because of the tissue's large dependence on mitochondrial ATP provision. Our study revealed in aged rat heart a significant 25% decrease in mtDNA relative content, a significant 29% increase in the 4.8 Kb mtDNA deletion relative content, and a significant inverse correlation between such contents as well as a significant 38% decrease in TFAM protein amount. The TFAM-binding activity to specific mtDNA regions increased at those encompassing the mtDNA replication origins, D-loop and Ori-L. The same mtDNA regions were screened for different kinds of oxidative damage, namely Single Strand Breaks (SSBs), Double Strand Breaks (DSBs), abasic sites (AP sites) and oxidized bases as 7,8-dihydro-8-oxoguanine (8oxoG). A marked increase in the relative content of mtDNA strand damage (SSBs, DSBs and AP sites) was found in the D-loop and Ori-L regions in the aged animals, unveiling for the first time in vivo an age-related, non-stochastic accumulation of oxidative lesions in these two regions that appear as hot spots of mtDNA damage. The use of Formamidopyrimidine glycosylase (Fpg) demonstrated also a significant age-related accumulation of oxidized purines particularly in the D-loop and Ori-L regions. The detected increased binding of TFAM to the mtDNA damage hot spots in aged heart suggests a link between TFAM binding to mtDNA and loss of mitochondrial genome likely through hindrance of repair processes.

Optimised detection of mitochondrial DNA strand breaks

Intrinsic and extrinsic factors that induce cellular oxidative stress damage tissue integrity and promote ageing, resulting in accumulative strand breaks to the mitochondrial DNA (mtDNA) genome. Limited repair mechanisms and close proximity to superoxide generation make mtDNA a prominent biomarker of oxidative damage. Using human DNA we describe an optimised long-range qPCR methodology that sensitively detects mtDNA strand breaks relative to a suite of short mitochondrial and nuclear DNA housekeeping amplicons, which control for any variation in mtDNA copy number. An application is demonstrated by detecting 16-36-fold mtDNA damage in human skin cells induced by hydrogen peroxide and solar simulated radiation.

Low-level mitochondrial heteroplasmy modulates DNA replication, glucose metabolism and lifespan in mice

Mutations in mitochondrial DNA (mtDNA) lead to heteroplasmy, i.e., the intracellular coexistence of wild-type and mutant mtDNA strands, which impact a wide spectrum of diseases but also physiological processes, including endurance exercise performance in athletes. However, the phenotypic consequences of limited levels of naturally arising heteroplasmy have not been experimentally studied to date. We hence generated a conplastic mouse strain carrying the mitochondrial genome of an AKR/J mouse strain (B6-mtAKR) in a C57BL/6 J nuclear genomic background, leading to >20% heteroplasmy in the origin of light-strand DNA replication (OriL). These conplastic mice demonstrate a shorter lifespan as well as dysregulation of multiple metabolic pathways, culminating in impaired glucose metabolism, compared to that of wild-type C57BL/6 J mice carrying lower levels of heteroplasmy. Our results indicate that physiologically relevant differences in mtDNA heteroplasmy levels at a single, functionally important site impair the metabolic health and lifespan in mice.

Human Mitochondrial Transcription Initiation Complexes Have Similar Topology on the Light and Heavy Strand Promoters

Transcription is a highly regulated process in all domains of life. In human mitochondria, transcription of the circular genome involves only two promoters, called light strand promoter (LSP) and heavy strand promoter (HSP), located in the opposite DNA strands. Initiation of transcription occurs upon sequential assembly of an initiation complex that includes mitochondrial RNA polymerase (mtRNAP) and the initiation factors mitochondrial transcription factor A (TFAM) and TFB2M. It has been recently suggested that the transcription initiation factor TFAM binds to HSP and LSP in opposite directions, implying that the mechanisms of transcription initiation are drastically dissimilar at these promoters. In contrast, we found that binding of TFAM to HSP and the subsequent recruitment of mtRNAP results in a pre-initiation complex that is remarkably similar in topology and properties to that formed at the LSP promoter. Our data suggest that assembly of the pre-initiation complexes on LSP and HSP brings these transcription units in close proximity, providing an opportunity for regulatory proteins to simultaneously control transcription initiation in both mtDNA strands.

Mitochondrial DNA as a biosensor of UV exposure in human skin.

Mitochondrial DNA (mtDNA) has been demonstrated to be a reliable biomarker of UV-induced genetic damage in both animal and human skin. Properties of the mitochondrial genome which allow for its use as a biomarker of damage include its presence in multiple copies within a cell, its limited repair mechanisms, and its lack of protective histones. To measure UV-induced mtDNA damage (particularly in the form of strand breaks), real-time quantitative PCR (qPCR) is used, based on the observation that PCR amplification efficiency is decreased in the presence of high levels of damage. Here, we describe the measurement of UV-induced mtDNA damage, including the extraction of cellular DNA, qPCR to determine the relative amount of mtDNA, qPCR to determine UV-induced damage within a long strand of mtDNA, and the verification of the amplification process using gel electrophoresis.

Functional Genomic Analysis of Human Mitochondrial RNA Processing

Both strands of human mtDNA are transcribed in continuous, multigenic units that are cleaved into the mature rRNAs, tRNAs, and mRNAs required forrespiratory chain biogenesis. We sought to systematically identify nuclear-encoded proteins that contribute to processing of mtRNAs within the organelle. First, we devised and validated a multiplex MitoString assay that quantitates 27 mature and precursor mtDNA transcripts. Second, we applied MitoString profiling to evaluate the impact of silencing each of 107 mitochondrial-localized, predicted RNA-binding proteins. With the resulting data set, we rediscovered the roles of recently identified RNA-processing enzymes, detected unanticipated roles of known disease genes in RNA processing, and identified new regulatory factors. We demonstrate that one such factor, FASTKD4, modulates the half-lives of a subset of mt-mRNAs and associates with mtRNAs invivo. MitoString profiling may be useful for diagnosing and deciphering the pathogenesis of mtDNA disorders.