Mitochondrion

Mitochondria are dynamic organelles that play crucial roles in energy transformation, biosynthesis, and cellular signaling. They actively process biological information, detecting and reacting to both internal and external stimuli. Through intricate physical interactions and diffusion mechanisms within cellular networks, mitochondria integrate diverse inputs and generate signals that finely adjust cellular functions and overall physiology. As a result, the phenotypic expressions of impaired mitochondrial function can exhibit high variability. High-throughput transcriptomic data can capture these changes, but traditional pathway analyses performed on common databases often struggle to specifically detect mitochondrial alterations. This is primarily due to the size of pathways, where the mitochondrial component is typically a small portion of the examined signaling network. To allow a specific exploration of mitochondrial activity through transcriptomic profiles, we developed the mitology R package. We started with a collection of genes whose proteins localize in to the mitochondria, derived from specialized databases like MitoCarta, IMPI, MSeqDR, and from the Gene Ontology database (genes annotated with terms related to mitochondri- in their description). Then, exploiting the mitochondrial gene list, mitology provides ready-to-use implementations of MitoCarta pathways and also a reorganization of general pathway databases, like Reactome and Gene Ontology, with a specific focus on the pathways related to mitochondria activity. Finally, mitology utilizes these mitochondria-focused pathways to conduct mitochondrial pathway analyses, enabling single-sample assessments. Furthermore, we extended the functionality of our package to accommodate classical gene expression data, as well as the newest techniques of single-cell and spatial transcriptomics. Mitology emerges as a novel R package adept at dissecting and unraveling mitochondrial activity, serving as an instrument for conducting targeted mitochondrial studies.

Strictly maternal inheritance and lack of intermolecular recombination of the human mitochondrial genome (mtDNA) are the assumed preconditions for molecular evolution studies, phylogenetic reconstruction and population genetic analyses. This hypothesis, however, has been challenged by investigations providing evidence for genetic recombination of mtDNA, thus sparking controversy. Using single-molecule real-time (SMRT) sequencing technology, we sequenced the entire mtDNA from blood and fibroblast cells from five individuals with biparental mtDNA transmission in three separate, multiple-generation families. After phasing the single nucleotide polymorphism (SNP) genotypes of mtDNA, no intermolecular recombination between paternal and maternal mtDNA was found when the mtDNA was transmitted in either biparental or maternal mode. Our study provides support for the argument that intermolecular mtDNA recombination is absent or extremely rare in humans. As a consequence, these results support the feasibility of mtDNA-based molecular evolution studies and phylogenetic and population genetic analyses for humans, while also avoiding inaccurate phylogenetic inferences and incorrect rejection of the molecular clock.

Aging, characterized by a series of functional declines correlated with advancing chronological age, has a significant mitochondrial DNA (mtDNA) component, with somatic mtDNA deletions playing a central role. In post-mitotic or slow-dividing cells like neurons and skeletal muscles, selfish mtDNA deletions clonally expand within a cell, ultimately leading to the deterioration and death of host cells and appearence of age-related phenotypes. Thus reducing the burden of somatic deletions could have far- reaching systemic benefits for the entire human body. Given the crucial role of direct nucleotide repeats in the formation of mitochondrial deletions, we hypothesize that minimizing these repeats in the human mitochondrial genome could enhance healthspan by decreasing somatic deletions. To investigate this hypothesis, we focus on the "common repeat", a 13-base pair perfect direct repeat sequence (ACCTCCCTCACCA) located at positions 8470-8482 and 13447-13459, respectively. This perfect repeat: (i) is highly prevalent, with its potential deleterious consequences affecting the majority of humans; (ii) represents one of the most fragile sites, highly prone to forming deletions; (iii) when disrupted, is associated with a decreased somatic deletion load and enhanced human healthspan; (iv) is likely to experience positive selection in the present or near future due to indirect fitness effects, such as the "grandmother effect", and direct fitness effects, such as (v) a decreased mutation rate. These observations support the argument that reducing the mtDNA somatic deletion load through targeted disruption of these repeats, or by using naturally occurring polymorphisms with disrupted repeats in mitochondrial medicine, could be an effective approach to increasing human longevity.

Serrano et al. (Serrano et al., 2024) use a high-fidelity somatic mtDNA mutation analysis in conplastic mice in which mtDNA was replaced with exogenous mtDNA of different mouse strains. Serrano reported apparent abundant somatic reversion mutations in the exogenous mtDNA that seemed to restore the original mito-nuclear match. If real, such a phenomenon would have important implications for health and genetics. In todays highly mixed human population, the pairing of potentially mismatched nuclear and mitochondrial genomes is widespread, so the proposed reversion mutagenesis should be commonplace. We demonstrate, however, that these reversion mutations are not real but originate from cross-contamination between samples and from NUMTs, the mtDNA pseudogenes located in the nuclear genome.

Whether heteroplasmic mitochondrial genetic variants in readily accessible tissues (blood, skeletal muscle) reflect those in the human heart (atrial appendage, left ventricle) is unknown. Using next generation sequencing data from paired tissue samples (n=233) collected postmortem in the Genotype-Tissue Expression project, we identified 558 unique heteroplasmic mitochondrial genetic variants across the four tissues, of which only 13% were shared across all four tissue sites. Between the two cardiac sites, 61% of heteroplasmic mitochondrial genetic variants were unique to one site. A greater proportion of the heteroplasmic variants were non-synonymous or frameshift variants in the muscle sites compared to blood or those variants shared across all four tissues. Compared to blood, the total number of heteroplasmic variants was higher in cardiac tissue, which was associated with advancing age. Our findings suggest that human cardiac tissue has unique heteroplasmic mtDNA variants and may be relevant to aging-related diseases.

The mutational spectrum of the mitochondrial DNA (mtDNA) does not resemble any of the known mutational signatures of the nuclear genome and variation in mtDNA mutational spectra between different organisms is still incomprehensible. Since mitochondria is tightly involved in aerobic energy production, it is expected that mtDNA mutational spectra is affected by the oxidative damage. Assuming that oxidative damage increases with age, we analyze mtDNA mutagenesis of different species. Analysing (i) dozens thousands of somatic mtDNA mutations in samples of different age (ii) 70053 polymorphic synonymous mtDNA substitutions, reconstructed in 424 mammalian species with different generation length and (iii) synonymous nucleotide content of 650 complete mitochondrial genomes of mammalian species we observed that the frequency of AH>GH substitutions (H - heavy chain notation) is twice higher in species with high versus low generation length making their mtDNA more AH poor and GH rich. Considering that AH>GH substitutions are also sensitive to the time spent single stranded (TSSS) during asynchroniuos mtDNA replication we demonstrated that AH>GH substitution rate is a function of both species-specific generation length and position specific TSSS. We propose that AH>GH is a mitochondria-specific signature of oxidative damage associated with both aging and TSSS.

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.

Mitochondria are central to cellular function, particularly in metabolically active tissues such as skeletal muscle. Nuclear-encoded RNAs typically localise within the nucleus and cytosol but a small population may also translocate to subcellular compartments such as mitochondria. We aimed to investigate the nuclear-encoded RNAs that localise within the mitochondria of skeletal muscle cells and tissue. Intact mitochondria were isolated via immunoprecipitation (IP) followed by enzymatic treatments (RNase-A and proteinase-K) optimised to remove transcripts located exterior to mitochondria, making it amenable for high-throughput transcriptomic sequencing. Small-RNA sequencing libraries were successfully constructed from as little as 1.8 ng mitochondrial RNA input. Small-RNA sequencing of mitochondria from rat myoblasts revealed the enrichment of over 200 miRNAs. Whole-transcriptome RNA sequencing of enzymatically-purified mitochondria isolated by IP from skeletal muscle tissue showed a striking similarity in the degree of purity compared to mitoplast preparations which lack an outer mitochondrial membrane. In summary, we describe a novel, powerful sequencing approach applicable to animal and human tissues and cells that can facilitate the discovery of nuclear-encoded RNA transcripts localised within skeletal muscle mitochondria.

The resilience of the mitochondrial genome to a high mutational pressure depends, in part, on purifying selection against detrimental mutations in the germline. It is crucial to understand the mechanisms of this process. Recently, Floros et al. concluded that much of the purifying selection takes place during the proliferation of primordial germ cells (PGCs) because, according to their analysis, the synonymity of mutations in late PGCs was seemingly increased compared to those in early PGCs. We re-analyzed the Floros et al. mutational data and discovered a high proportion of sequence variants that are not true mutations, but originate from NUMTs, the latter of which are segments of mitochondrial DNA (mtDNA) inserted into nuclear DNA, up to millions of years ago. This is a well-known artifact in mtDNA mutational analysis. Removal of these artifacts from the Floros et al. dataset abolishes the reported effect of purifying selection in PGCs. We therefore conclude that the mechanism of germline selection of mtDNA mutations remains open for debate, and more research is needed to fully elucidate the timing and nature of this process.

MotivationTraditional methods for detecting large-scale mitochondrial DNA (mtDNA) deletions (LSMDs) in cells present challenges, i.e. a priori information, high DNA inputs, poor sensitivity and are not always quantitative. Mitigation can be achieved through high throughput DNA sequencing using e.g. Illumina and Oxford Nanopore Technologies (ONT), in combination with LSMD breakpoint identification and quantification using bioinformatic tools. Splice-aware RNA alignment tools increase the sensitivity for detecting LSMD breakpoints compared with DNA aligners. Long-read sequencing (LRS) also offers potential advantages over short read sequencing, e.g. greater read lengths and capturing variants on single reads. No existing pipelines capture the benefits of both a splice-aware alignment tool and LRS. ResultsWe developed "NanoDel", a LRS pipeline, to sensitively and accurately detect cellular LSMDs. Using artificial datasets, "NanoDel" was more sensitive and accurate than other pipelines. In samples diagnosed with mitochondrial disease, it identified both known and previously uncharacterised (including mixtures) of LSMDs, without a priori information. LSMD breakpoints were found in mt-co1, mt-cyb, mt-nd6 and mt-nd5 genes. Analysis of selected LSMDs revealed proximity to repeat and putative G-quadruplex motifs, and occurrence in a range of healthy and pathological tissues, indicating potential for a shared vulnerability landscape in mtDNA, shaped by sequence motifs and structural constraints. "NanoDel" combined with one-amplicon, not two-amplicon, LR-PCR offers a robust strategy with clinical application for detecting LSMDs across a variety of cell/tissue samples, and its application across a broader range of samples, will yield new mechanistic insights into LSMD formation, and further our understanding of mtDNA instability.

Mitochondrial DNA copy number (mtDNA-CN) is a proxy for mitochondrial function and has been of increasing interest to the mitochondrial research community. There are a number of ways to measure mtDNA-CN, ranging from qPCR to whole genome sequencing [1]. A recent article in the Journal of Molecular Diagnostics [2] described a novel method for measuring mtDNA-CN that is both inexpensive and reproducible. After adapting the assay for use in our lab, we have found it to be reproducible and well-correlated with mtDNA-CN derived from whole genome sequencing. However, certain individuals show poor concordance between the two measures, particularly individuals with qPCR mtDNA-CN measurements >3 standard deviations below the sample mean, which corresponds to roughly 1% of assayed individuals (Figure 1). After examining whole genome sequencing data, this seems to be due to specific polymorphisms within the D-loop primer region, at positions MT 338, 340, 452, 457, 458, 460, 461, 466, and 467. All individuals with a variant in at least one of these positions have non-concordant mtDNA-CN measurements. Meanwhile, variants observed at other positions within the primer region do not appear to cause dropout. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/463983v1_fig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@134ca51org.highwire.dtl.DTLVardef@ce9196org.highwire.dtl.DTLVardef@1b82af6org.highwire.dtl.DTLVardef@c9701_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Discrepancy between the monochrome multiplex qPCR mtDNA-CN and the whole genome sequencing mtDNA-CN for 1,732 distinct individuals. Data are centered at 0 and scaled so that the standard deviation = 1. The dotted red line represents 3 standard deviations beneath the sample mean. Individuals in the U, L1, L4, and T haplogroups have a disproportionately higher risk of discordant measures between the two assays. C_FIG

With very few exceptions, mitochondrial DNA (mtDNA) in humans is transmitted exclusively from mothers to their offspring, suggesting the presence of a strong evolutionary pressure favoring the exclusion of paternal mtDNA. We have recently shown strong evidence of paternal mtDNA transmission. In these rare situations, males exhibiting biparental mtDNA appear to be limited to transmitting just one of the mtDNA species to their offspring, while females possessing biparental mtDNA populations consistently transmit both populations to their offspring at a very similar heteroplasmy level. The precise biological and genetic factors underlying this unusual transmission event remain unclear. Here, we have examined heteroplasmy levels in various tissues among individuals with biparental inheritance. Our results indicate that individuals with biparental mtDNA have remarkable inter-tissue variability in heteroplasmy level. At the single-cell level, paternal mtDNA heteroplasmy in sperm varies dramatically, and many sperm possess only one of the two mtDNA populations originally in question. These results show a fundamental, parent-of-origin difference in how mtDNA molecules transmit and propagate. This helps explain how a single population of mtDNAs are transmitted from a father possessing two populations of mtDNA molecules, suggesting that some mtDNA populations may be favored over others when transmitted from the father.

In this work, we adapted a method for isolation of a highly purified fraction of mitochondrial DNA from muscle tissues suitable for further sample preparation of libraries without an amplification step for sequencing tasks on various NGS platforms and a method for evaluating the purity of DNA from contamination by nuclear genome regions. We optimized several techniques7,8 for enrichment of the mitochondrial fractions and purifying mtDNA. Here, we describe a protocol that allows getting from 80-100 mg of muscle tissues up to 1000 ng mtDNA, almost free from impurities of RNA and fragments of the nuclear genome.To assess the degree of purity of human mtDNA fraction from impurities of the nuclear genome, we adapted the PCR-screening technique7 for the beta-actin gene region and AluSx-repeats in the human genome. This methodology avoids false-heteroplasmy calls (PCR biases or NUMT contamination) that occur when long-range PCR amplification is used for mtDNA enrichment.

Mitochondrial DNA copy number (mtDNA-CN) is a metric of mitochondrial function that has been associated with a variety of diseases including cardiovascular disease and all-cause mortality. To investigate genes and pathways affected by mtDNA-CN variation, we perturbed HEK 293T cells with ethidium bromide to deplete mtDNA. Using RNASeq and methylation microarrays, we evaluated transcriptomic and methylomic changes in treated cell lines. We observed an 8-fold decrease in mtDNA-CN and compensatory shifts in mitochondrial transcription to support mtDNA replication. Nuclear transcriptomic and methylomic analysis highlighted changes in metabolic pathways, including oxidative phosphorylation and canonical glycolysis. Longitudinal analyses revealed that the identified genes and pathways have different response timing, with nuclear response lagging behind mitochondrial response. These findings further elucidate the mechanisms behind mtDNA maintenance and responses to cellular energetics as well as mitochondrial-nuclear crosstalk dynamics.

ii.Mitochondrial proteomes have been experimentally characterized for only a handful of animal species. However, the increasing availability of genomic and transcriptomic data allows one to infer mitochondrial proteins using computational tools. MitoPredictor is a novel random-forest classifier, which utilizes orthology-search, mitochondrial targeting signal (MTS) identification, and protein domain content to infer mitochondrial proteins in animals. MitoPredictors output also includes an easy-to-use R Shiny applet for the visualization and analysis of the results. In this article, we provide a guide for predicting and analyzing the mitochondrial proteome of the ctenophore Mnemiopsis leidyi using MitoPredictor.

Folate-mediated one-carbon metabolism (FOCM) is required for the biosynthesis of purines, thymidylate (dTMP), and methionine. Maintenance of adequate cellular dTMP levels is essential to preserve the integrity of the nuclear and mitochondrial genomes. Inadequate dTMP production leads to uracil misincorporation into mitochondrial DNA (mtDNA) and impaired mitochondrial function. However, the mechanisms whereby uracil in mtDNA impairs mitochondrial function are uncharacterized. The release of mtDNA into the cytosol acts as a damage-associated molecular pattern (DAMP), resulting in inflammation and cell death. Previously, we reported that disrupting mitochondrial dTMP synthesis through serine hydroxymethyltransferase 2 (Shmt2) heterozygosity increases uracil in mtDNA and impairs mitochondrial function in mice. This study aimed to investigate whether impairment of mitochondrial FOCM through Shmt2 disruption leads to the release of mtDNA into the cytosol. Shmt2+/- MEF cells showed a > 2-fold increase in cytosolic mtDNA leakage compared to Shmt2+/+ cells (p<0.05). There was no significant difference in total mtDNA content between Shmt2+/+ and Shmt2+/- MEF cells. MEFs with decreased Shmt2 expression activated apoptosis by the ratio of cleaved caspase-3 to caspase-3. In addition, SHMT2 knock-out (SHMT2 KO) haploid chronic myeloid leukemia (HAP1) cells also exhibited increased cytosolic mtDNA content compared to the wild-type (WT) HAP1 cells. HAP1 lacking Shmt2 expression activated the cGAS/STING pathway, but suppressed apoptosis, compared to WT HAP1 cells. This study demonstrates that decreased Shmt2 expression leads to cytosolic mtDNA leakage and that the downstream effects of mtDNA leakage vary by cell type.

Mitochondria are essential organelles present in the vast majority of eukaryotic cells. Their central function is to produce cellular energy through the OXPHOS system, and functional alterations provoke so-called mitochondrial OXPHOS diseases. It is estimated that several hundred mitochondrial proteins have unknown functions. Very recently, C6orf203 was described to participate in mitochondrial transcription under induced mitochondrial DNA depletion stress conditions. Here, we describe another role for C6orf203, specifically in OXPHOS biogenesis under regular culture conditions. HEK293T C6orf203-Knockout (KO) cells generated by CRISPR/Cas9 genome editing showed both reduced grow in galactose, as a carbon source, and in their oxygen consumption capability, strongly suggesting an OXPHOS dysfunction. C6orf203-KO also provoked a depletion of OXPHOS proteins and decreased the activity of the mitochondrial respiratory chain complexes. C6orf203 was present in high molecular weight complexes compatible with mitoribosomes, and in vivo labelling of de novo mitochondrial proteins synthesis revealed that C6orf203-KO severely but not completely affected the translation of mitochondrial mRNAs. Taken together, we describe herein a new function for C6orf203, making it a potential OXPHOS disease-related candidate.

Recent evidence suggests that the coding potential of the mitogenome is underestimated. We found a downstream alternative ATG initiation codon in the +3 reading frame of the human mitochondrial nd4 gene. This newly characterized alternative open reading frame (altORF) encodes a 99-amino acids long polypeptide, MTALTND4, which is conserved in primates. This small protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts mitochondrial physiology. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases.

BackgroundPrediction of pathogenic variants is one of the biggest challenges for researchers and clinicians in the time of next-generation sequencing technologies. Stratification of individuals based on truly pathogenic variants might lead to improved, personalized treatments.\n\nResultsWe present Frequency Conservation Score (FCS) and Frequency Conservation Score for Mitochondrial DNA (FCSMt) two methods for the detection of pathogenic single nucleotide variants in nuclear and mitochondrial DNA, respectively. These scores are based in a random forest model trained over a set of potentially relevant predictors: (i) conservation scores (PhastCons and phyloP); (ii) locus variability at each genomic position built from gnomAD database and (iii) physicochemical distance for amino acids substitutions and the impact/consequence over the canonical transcript. FCS showed an AUC of 98% for deleteriousness in an independent validation dataset, outperforming other scores such as metaLR, metaSVM, REVEL, DANN, CADD, SIFT, PROVEAN or FATHMM-MKL. Moreover, FCSMt presented an AUC=0.92 for pathogenic mitochondrial SNVs detection. The tool is available at http://bioinfo.cnic.es/FCS\n\nConclusionsFCS and FCS-Mt improve pathogenic mutation detection, allowing the prioritization of relevant variants in Whole Exome and Whole Genome Sequencing Analysis.

Every cell in our body contains a vibrant population of mitochondria, or, more precisely, of mitochondrial DNA molecules (mtDNAs). Just like members of any population mtDNAs multiply (by replication) and die (i.e., are removed, either by degradation or by distribution into the sister cell in mitosis). An intriguing question is whether all mitochondria in this population are equal, especially whether some are responsible primarily for reproduction and some - for empowering the various jobs of the mitochondrion, oxidative phosphorylation in the first place. Importantly, because mtDNA is highly damaged such a separation of responsibilities could help greatly reduce the conversion of DNA damage into real inheritable mutations. An unexpected twist in the resolution of this problem has been brought about by a recent high-precision analysis of mtDNA mutations (Sanchez-Contreras et al. 2023). They discovered that certain transversion mutations, unlike more common transitions, are not accumulating with age in mice. We argue that this observation requires the existence of a permanent replicating subpopulation/lineage of mtDNA molecules, which are protected from DNA damage, a.k.a. the stem mtDNA. This also implies the existence of its antipode i.e., the worker mtDNA, which empowers OSPHOS, sustains damage and rarely replicates. The analysis of long HiFi reads of mtDNA performed by PacBio closed circular sequencing confirms this assertion.