Mammalian Mitochondrial Genome Research Articles

Illuminating mitochondrial translation through mouse models.

Mitochondria are hubs of metabolic activity with a major role in ATP conversion by oxidative phosphorylation (OXPHOS). The mammalian mitochondrial genome encodes 11 mRNAs encoding 13 OXPHOS proteins along with 2 rRNAs and 22 tRNAs, that facilitate their translation on mitoribosomes. Maintaining the internal production of core OXPHOS subunits requires modulation of the mitochondrial capacity to match the cellular requirements and correct insertion of particularly hydrophobic proteins into the inner mitochondrial membrane. The mitochondrial translation system is essential for energy production and defects result in severe, phenotypically diverse diseases, including mitochondrial diseases that typically affect postmitotic tissues with high metabolic demands. Understanding the complex mechanisms that underlie the pathologies of diseases involving impaired mitochondrial translation is key to tailoring specific treatments and effectively targeting the affected organs. Disease mutations have provided a fundamental, yet limited, understanding of mitochondrial protein synthesis, since effective modification of the mitochondrial genome has proven challenging. However, advances in next generation sequencing, cryoelectron microscopy, and multi-omic technologies have revealed unexpected and unusual features of the mitochondrial protein synthesis machinery in the last decade. Genome editing tools have generated unique models that have accelerated our mechanistic understanding of mitochondrial translation and its physiological importance. Here we review the most recent mouse models of disease pathogenesis caused by defects in mitochondrial protein synthesis and discuss their value for preclinical research and therapeutic development.

Rapid molecular species identification of mammalian scat samples using nanopore adaptive sampling

Rapid molecular species identification of mammalian scat samples using nanopore adaptive sampling

Mammal mitogenomics from invertebrate‐derived DNA

AbstractThe metabarcoding of vertebrate DNA found in invertebrate‐derived DNA (iDNA) has proven a powerful tool for monitoring biodiversity. To date, iDNA has primarily been used to detect the presence/absence of particular taxa using metabarcoding, though recent efforts demonstrated the potential utility of these data for estimating relative animal abundance. Here, we test whether iDNA can also be used to reconstruct complete mammalian mitogenomes and therefore bring the field closer to population‐level analyses. Specifically, we used mitogenomic hybridization capture coupled with high‐throughput sequencing to analyze individual (N = 7) or pooled (N = 5) fly‐derived DNA extracts, and individual (N = 7) or pooled (N = 1) leech‐derived DNA extracts, which were known a priori to contain primate DNA. All sources of iDNA showed their ability to generate large amounts of mammalian mitogenomic information and deeper sequencing of libraries is predicted to allow for even more complete recovery of primate mitogenomes from most samples (90%). Sixty percent of these iDNA extracts allowed for the recovery of (near) complete mammalian mitochondrial genomes (hereafter mitogenomes) that proved useable for phylogenomic analyses. These findings contribute to paving the way for iDNA‐based population mitogenomic studies of terrestrial mammals.

Organization and expression of the mammalian mitochondrial genome.

The mitochondrial genome encodes core subunits of the respiratory chain that drives oxidative phosphorylation and is, therefore, essential for energy conversion. Advances in high-throughput sequencing technologies and cryoelectron microscopy have shed light on the structure and organization of the mitochondrial genome and revealed unique mechanisms of mitochondrial gene regulation. New animal models of impaired mitochondrial protein synthesis have shown how the coordinated regulation of the cytoplasmic and mitochondrial translation machineries ensures the correct assembly of the respiratory chain complexes. These new technologies and disease models are providing a deeper understanding of mitochondrial genome organization and expression and of the diseases caused by impaired energy conversion, including mitochondrial, neurodegenerative, cardiovascular and metabolic diseases. They also provide avenues for the development of treatments for these conditions.

A Synthetic Small RNA Homologous to the D-Loop Transcript of mtDNA Enhances Mitochondrial Bioenergetics.

Mitochondrial malfunction is a hallmark of many diseases, including neurodegenerative disorders, cardiovascular and lung diseases, and cancers. We previously found that alveolar progenitor cells, which are more resistant to cigarette smoke-induced injury than the other cells of the lung parenchyma, upregulate the mtDNA-encoded small non-coding RNA mito-ncR-805 after exposure to smoke. The mito-ncR-805 acts as a retrograde signal between the mitochondria and the nucleus. Here, we identified a region of mito-ncR-805 that is conserved in the mammalian mitochondrial genomes and generated shorter versions of mouse and human transcripts (mmu-CR805 and hsa-LDL1, respectively), which differ in a few nucleotides and which we refer to as the “functional bit”. Overexpression of mouse and human functional bits in either the mouse or the human lung epithelial cells led to an increase in the activity of the Krebs cycle and oxidative phosphorylation, stabilized the mitochondrial potential, conferred faster cell division, and lowered the levels of proapoptotic pseudokinase, TRIB3. Both oligos, mmu-CR805 and hsa-LDL1 conferred cross-species beneficial effects. Our data indicate a high degree of evolutionary conservation of retrograde signaling via a functional bit of the D-loop transcript, mito-ncR-805, in the mammals. This emphasizes the importance of the pathway and suggests a potential to develop this functional bit into a therapeutic agent that enhances mitochondrial bioenergetics.

R-Loops and Mitochondrial DNA Metabolism.

R-loops forming inadvertently during transcription can threaten genome stability, but R-loops are also formed intentionally, as a means of regulating transcription and other aspects of DNA metabolism. The study of R-loops in mitochondria is in its infancy, and yet there is already clear evidence that they are predominantly located in the major regulatory region of the mammalianmitochondrial genome. Here, we describe how mitochondrial R-loops have been characterized to date, with the emphasis on the problems of their being extremely labile, and how to minimize their loss during extraction. The oft-overlooked issues of RNA-DNA hybrids not being synonymous with R-loops, and adventitious RNA hybridization to DNA, are tackled head on; and possible new approaches are described and placed in the context of future research lines that could reveal the detailed roles of R-loops in the metabolism of mitochondrial DNA.

Malignancy and NF-\u03baB signalling strengthen coordination between expression of mitochondrial and nuclear-encoded oxidative phosphorylation genes

BackgroundMitochondria are ancient endosymbiotic organelles crucial to eukaryotic growth and metabolism. The mammalian mitochondrial genome encodes for 13 mitochondrial proteins, and the remaining mitochondrial proteins are encoded by the nuclear genome. Little is known about how coordination between the expression of the two sets of genes is achieved.ResultsCorrelation analysis of RNA-seq expression data from large publicly available datasets is a common method to leverage genetic diversity to infer gene co-expression modules. Here we use this method to investigate nuclear-mitochondrial gene expression coordination. We identify a pitfall in correlation analysis that results from the large variation in the proportion of transcripts from the mitochondrial genome in RNA-seq data. Commonly used normalisation techniques based on total read counts, such as FPKM or TPM, produce artefactual negative correlations between mitochondrial- and nuclear-encoded transcripts. This also results in artefactual correlations between pairs of nuclear-encoded genes, with important consequences for inferring co-expression modules beyond mitochondria. We show that these effects can be overcome by normalizing using the median-ratio normalisation (MRN) or trimmed mean of M values (TMM) methods. Using these normalisations, we find only weak and inconsistent correlations between mitochondrial and nuclear-encoded mitochondrial genes in the majority of healthy human tissues from the GTEx database.ConclusionsWe show that a subset of healthy tissues with high expression of NF-κB show significant coordination, suggesting a role for NF-κB in ensuring balanced expression between mitochondrial and nuclear genes. Contrastingly, most cancer types show robust coordination of nuclear and mitochondrial OXPHOS gene expression, identifying this as a feature of gene regulation in cancer.

RNA Crosslinking to Analyze the Mitochondrial RNA-Binding Proteome.

Even though the mammalian mitochondrial genome (mtDNA) is very small and only codes for 13 proteins, all being subunits of the oxidative phosphorylation system, it requires several hundred nuclear encoded proteins for its maintenance and expression. These include replication and transcription factors, approximately 80 mitoribosomal proteins and many proteins involved in the posttranscriptional modification, processing, and stability of mitochondrial RNAs. In recent years, many of these factors have been identified and functionally characterized, but the complete mtRNA-interacting proteome is not firmly established. Shotgun proteomics has been used successfully to define whole-cell polyadenylated RNA (poly(A)-RNA) interacting proteomes using the nucleotide analogue 4-thiouridine (4SU) combined with UV crosslinking, poly(A)-RNA isolation and mass spectrometry to identify all poly(A)-RNA bound proteins. Although in this case also a considerable number of mitochondrial proteins were identified, the method was not specifically directed at the mitochondrial poly(A)-RNA bound proteome. Here we describe a method for enrichment of the mitochondrial poly(A)-RNA bound proteome based on 4SU labeling and UV crosslinking. The method can be applied either for isolated mitochondria prior to UV crosslinking or for whole-cell crosslinking followed by mitochondrial isolation.

Complete mitochondrial genome of red panda (Ailurus fulgens) and its phylogenetic analysis

In this study, we reported the complete mitochondrial genome sequence of a red panda (Ailurus fulgens). The mitogenome (GenBank accession number MK886830) length is 16,517 bp and exhibits the typical structure of mammalian mitochondrial genomes contains 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and one control region. The tRNASer (AGY) gene failed to form the typical clover-leaf secondary structure as it lacked the dihydrouridine (DHU) arm. phylogenetically, our sequence cluster together with genus Ailurus, which showed a closer genetic relationship. The mitogenome provides new data to further elucidation and understand the phylogeny of red pandas.

Twist and Turn-Topoisomerase Functions in Mitochondrial DNA Maintenance.

Like any genome, mitochondrial DNA (mtDNA) also requires the action of topoisomerases to resolve topological problems in its maintenance, but for a long time, little was known about mitochondrial topoisomerases. The last years have brought a closer insight into the function of these fascinating enzymes in mtDNA topology regulation, replication, transcription, and segregation. Here, we summarize the current knowledge about mitochondrial topoisomerases, paying special attention to mammalian mitochondrial genome maintenance. We also discuss the open gaps in the existing knowledge of mtDNA topology control and the potential involvement of mitochondrial topoisomerases in human pathologies. While Top1mt, the only exclusively mitochondrial topoisomerase in mammals, has been studied intensively for nearly a decade, only recent studies have shed some light onto the mitochondrial function of Top2β and Top3α, enzymes that are shared between nucleus and mitochondria. Top3α mediates the segregation of freshly replicated mtDNA molecules, and its dysfunction leads to mtDNA aggregation and copy number depletion in patients. Top2β, in contrast, regulates mitochondrial DNA replication and transcription through the alteration of mtDNA topology, a fact that should be acknowledged due to the frequent use of Topoisomerase 2 inhibitors in medical therapy.

Ultracentrifugation enrichment protocol followed by total RNA sequencing allows assembly of the complete mitochondrial genome

The mitochondrial genome is an independent genetic system in each eukaryotic cell outside the nuclear genome. Mitochondrial DNA (mtDNA) appears in high copy number within one cell, unlike nuclear DNA, which exists in two copies. But nevertheless, mtDNA represent only small part of total cellular DNA what causes problematic analysis and identification of relevant mutations. While most researchers tend to overlook it because of its small size, the mitochondrial genome contains genes that are essential for cellular energetics and survival. Because of the increased awareness on the importance of metabolism and bioenergetics in a wide variety of human diseases, more and more mtDNA studies were performed. Mitochondrial genome research has established the connection between mtDNA and a wide variety of diseases such as cancer or neurodegenerative disorders. At the present time, several methods are known, that allow sequencing of mtDNA. However, genomic analysis is often complicated due to the low content of mtDNA compared to nuclear DNA. For this reason, we have designed a new approach to obtaining the genomic mitochondrial sequence. We chose RNA based sequencing. Since human mtDNA does not contain introns, the reconstruction of whole mitochondrial genome through RNA sequencing seems to be effective. Our method is based on total RNA sequencing coupled with simple ultracentrifugation protocol and de novo assembly. Following our protocol, we were able to assemble a complete mammalian mitochondrial genome with a length of 16,505 bp and an average coverage of 156. The method is a relatively simple and inexpensive which could help in the further research or diagnostics of mtDNA-based diseases.

Intramitochondrial transfer and engineering of mammalian mitochondrial genomes in yeast

Mitochondrial genomes (mtDNA) depend on the nuclear genome with which they have evolved to provide essential replication functions and have been known to replicate as xenotransplants only in the cells of closely related species. We now report that complete mouse mitochondrial genomes can be stably transplanted into the mitochondrial network in yeast devoid of their own mtDNA. Our analyses of these xenomitochondrial yeast cells show that they are accurately replicating intact mouse mtDNA genomes without rearrangement and that these mtDNA genomes have the same overall topology as the mtDNA present in the mouse mitochondrial network (i.e., circular monomers). Moreover, non-mtDNA replication and selection sequences required for maintaining the mitochondrial genomes in bacterial hosts are dispensable in these yeast mitochondria and could be efficiently and seamlessly removed by targeted homologous recombination within the mitochondria. These findings demonstrate that the yeast mtDNA replication system is capable of accurately replicating intact mammalian mtDNA genomes without sequence loss or rearrangement and that yeast mitochondria are a highly versatile host system for engineering complete mammalian mitochondrial genomes.

Gene annotation errors are common in the mammalian mitochondrial genomes database

BackgroundAlthough animal mitochondrial DNA sequences are known to evolve rapidly, their gene arrangements often remain unchanged over long periods of evolutionary time. Therefore, comparisons of mitochondrial genomes may result in significant insights into the evolution both of organisms and of genomes. Mammalian mitochondrial genomes recently published in the GenBank database of NCBI show numerous rearrangements in various regions of the genome, from which it may be inferred that the mammalian mitochondrial genome is more dynamic than expected. However, it is alternatively possible that these are errors of annotation and, if so, are misleading our interpretations. In order to verify these possible errors of annotation, we performed a comparative genomic analysis of mammalian mitochondrial genomes available in the NCBI database.ResultsUsing a combination of bioinformatics methods to carefully examine the mitochondrial gene arrangements in 304 mammalian species, we determined that there are only two sets of gene arrangements, one that is shared by all of the marsupials and another that is shared by all of the monotremes and eutherians, with these two arrangements differing only by the positions of tRNA genes in the region commonly designated as “WANCY” for the genes it comprises. All of the 68 other cases of reported gene rearrangements are errors. We note that there are also numerous errors of impossibly short, incorrect gene annotations, cases where genomes that are reported as complete are actually missing portions of the sequence, and genes that are clearly present but were not annotated in these records.ConclusionsWe judge that the application of simple bioinformatic tools in the verification of gene annotation, particularly for organelle genomes, would be a very useful enhancement for the curation of genome sequences submitted to GenBank.

MtDNA Chromatin-like Organization Is Gradually Established during Mammalian Embryogenesis

SummaryUnlike the nuclear genome, the mammalian mitochondrial genome (mtDNA) is thought to be coated solely by mitochondrial transcription factor A (TFAM), whose binding sequence preferences are debated. Therefore, higher-order mtDNA organization is considered much less regulated than both the bacterial nucleoid and the nuclear chromatin. However, our recently identified conserved DNase footprinting pattern in human mtDNA, which co-localizes with regulatory elements and responds to physiological conditions, likely reflects a structured higher-order mtDNA organization. We hypothesized that this pattern emerges during embryogenesis. To test this hypothesis, we analyzed assay for transposase-accessible chromatin sequencing (ATAC-seq) results collected during the course of mouse and human early embryogenesis. Our results reveal, for the first time, a gradual and dynamic emergence of the adult mtDNA footprinting pattern during embryogenesis of both mammals. Taken together, our findings suggest that the structured adult chromatin-like mtDNA organization is gradually formed during mammalian embryogenesis.

The complete mitochondrial genome of a blue sheep (Pseudois nayaur szechuanensis) from Gongga mountains of Sichuan, China

The blue sheep (Pseudois nayaur) belongs to the subfamily Caprinae. Here, we report the complete mitochondrial genome of a blue sheep subspecies (Pseudois nayaur szechuanensis) from Gongga Mountains of Sichuan, China. The genome (GenBank accession number MH719216) has a length of 16,664 bp and exhibits the typical structure of mammalian mitochondrial genomes. Phylogenetically, the newly generated mitochondrial genome is nested within the genus Pseudois group and formed a strongly supported (100% bootstrap) sister lineage to the previously reported P. n. szechuanensis (GenBank accession number, KJ784494). These results provide more molecular data for the genetic diversity conservation of this rare species.

Complete mitochondrial genome of a dwarf blue sheep (Pseudois schaeferi) and its phylogenetic analysis

The dwarf blue sheep (Pseudois schaeferi) belongs to the subfamily Caprinae within the Cetartiodactyla. Here, we report on the complete mitochondrial genome of a dwarf blue sheep from Nangqian of Qinghai, China. The genome (MK087727) has a length of 16,709 bp and exhibits the typical structure of mammalian mitochondrial genomes. Phylogenetically, the new dwarf blue sheep formed a strongly supported sister lineage to the previously reported P. schaeferi (JQ040802). These results support for dwarf blue sheep as a subspecies of Pseudois nayaur and provide more molecular data for conservation of the endemic species.

A New Numerical Method for DNA Sequence Analysis Based on 8-Dimensional Vector Representation

Background: The multiple sequence alignment (MSA) algorithms are the traditional ways to compare and analyze DNA sequences. However, for large DNA sequences, these algorithms require a long time computationally. Objective: Here we will propose a new numerical method to characterize and compare DNA sequences quickly. Method: Based on a new 2-dimensional (2D) graphical representation of DNA sequences, we can obtain an 8-dimensional vector using two basic concepts of probability, the mean and the variance. Results: We perform similarity/dissimilarity analyses among two real DNA data sets, the coding sequences of the first exon of beta-globin gene of 11 species and 31 mammalian mitochondrial genomes, respectively. Conclusion: Our results are in agreement with the existing analyses in our literatures. We also compare our approach with other methods and find that ours is more effective.

Novel Lines of Evidence for the Asymmetric Strand Displacement Model of Mitochondrial DNA Replication.

The mitochondrial genome, which consists of 16,569 bp of DNA with a cytosine-rich light (L) strand and a heavy (H) strand, exists as a multicopy closed circular genome within the mitochondrial matrix. The machinery for replication of the mammalian mitochondrial genome is distinct from that for replication of the nuclear genome. Three models have been proposed for mitochondrial DNA (mtDNA) replication, and one of the key differences among them is whether extensive single-stranded regions exist on the H strand. Here, three different methods that can detect single-stranded DNA (ssDNA) are utilized to identify the presence, location, and abundance of ssDNA on mtDNA. Importantly, none of these newly described methods involve the complication of prior mtDNA fractionation. The H strand was found to have extensive single-stranded regions with a profile consistent with the strand displacement model of mtDNA replication, whereas single strandedness was predominantly absent on the L strand. These findings are consistent with the in vivo occupancy of mitochondrial single-stranded DNA binding protein reported previously and provide strong new qualitative and quantitative evidence for the asymmetric strand displacement model of mtDNA replication.

Complete mitochondrial genome of an olive baboon (Papio anubis) from Gombe National Park, Tanzania

The olive baboon (Papio anubis) is the most widely distributed baboon species. We report here on the complete mitochondrial genome of an olive baboon from the south-eastern edge of the species’ range from Gombe National Park (NP), Tanzania. The genome (GenBank accession number MG787545) has a length of 16,490 bp and exhibits the typical structure of mammalian mitochondrial genomes. Phylogenetically, the olive baboon from Gombe NP is most closely related to eastern P. anubis, northern P. cynocephalus and P. hamadryas. The data are an important addition to further clarify the phylogeography of baboons and phylogeny of papionins in general.

Complete mitochondrial genome of a Toque Macaque (Macaca sinica)

The Toque macaque (Macaca sinica) is the only macaque species on Sri Lanka and endemic to the island. The newly generated mitochondrial genome (Genbank accession number MG870385), obtained from a captive individual, has a length of 16,525 bp and exhibits the typical structure of mammalian mitochondrial genomes. Phylogenetically, the Toque macaque is nested within the Macaca sinica species group and represents a sister lineage to a M. assamensis/M. thibetana clade. The new data help to further illuminate and better understand the complex phylogeny of macaques.