Reanalysis of mtDNA mutations of human primordial germ cells (PGCs) reveals NUMT contamination and suggests that selection in PGCs may be positive.

The resilience of the mitochondrial genome (mtDNA) to a high mutational pressure depends, in part, on negative purifying selection in the germline. A paradigm in the field has been that such selection, at least in part, takes place in primordial germ cells (PGCs). Specifically, Floros et al. (Nature Cell Biology 20: 144-51) reported an increase in the synonymity of mtDNA mutations (a sign of purifying selection) between early-stage and late-stage PGCs. We re-analyzed Floros' et al. data and determined that their mutational dataset was significantly contaminated with single nucleotide variants (SNVs) derived from a nuclear sequence of mtDNA origin (NUMT) located on chromosome 5. Contamination was caused by co-amplification of the NUMT sequence by cross-specific PCR primers. Importantly, when we removed NUMT-derived SNVs, the evidence of purifying selection was abolished. In addition to bulk PGCs, Floros et al. reported the analysis of single-cell late-stage PGCs, which were amplified with different sets of PCR primers that cannot amplify the NUMT sequence. Accordingly, there were no NUMT-derived SNVs among single PGC mutations. Interestingly, single PGC mutations show adecreaseof synonymity with increased intracellular mutant fraction. More specifically, nonsynonymous mutations show faster intracellular genetic drift towards higher mutant fraction than synonymous ones. This pattern is incompatible with predominantly negative selection. This suggests that germline selection of mtDNA mutations is a complex phenomenon and that the part of this process that takes place in PGCs may be predominantly positive. However counterintuitive, positive germline selection of detrimental mtDNA mutations has been reported previously andpotentially may be evolutionarily advantageous.

A mitochondria-specific mutational signature of aging: increased rate of A > G substitutions on the heavy strand.

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 are responsible for aerobic respiration, it is expected that mtDNA mutational spectrum is affected by oxidative damage. Assuming that oxidative damage increases with age, we analyse mtDNA mutagenesis of different species in regards to their generation length. Analysing, (i) dozens of thousands of somatic mtDNA mutations in samples of different ages (ii) 70053 polymorphic synonymous mtDNA substitutions reconstructed in 424 mammalian species with different generation lengths and (iii) synonymous nucleotide content of 650 complete mitochondrial genomes of mammalian species we observed that the frequency of AH > GH substitutions (H: heavy strand notation) is twice bigger 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 asynchronous 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.

Mitochondrial Pseudogenes Suggest Repeated Inter-Species Hybridization among Direct Human Ancestors.

The hypothesis that the evolution of humans involves hybridization between diverged species has been actively debated in recent years. We present the following novel evidence in support of this hypothesis: the analysis of nuclear pseudogenes of mtDNA ("NUMTs"). NUMTs are considered "mtDNA fossils" as they preserve sequences of ancient mtDNA and thus carry unique information about ancestral populations. Our comparison of a NUMT sequence shared by humans, chimpanzees, and gorillas with their mtDNAs implies that, around the time of divergence between humans and chimpanzees, our evolutionary history involved the interbreeding of individuals whose mtDNA had diverged as much as ~4.5 Myr prior. This large divergence suggests a distant interspecies hybridization. Additionally, analysis of two other NUMTs suggests that such events occur repeatedly. Our findings suggest a complex pattern of speciation in primate/human ancestors and provide one potential explanation for the mosaic nature of fossil morphology found at the emergence of the hominin lineage. A preliminary version of this manuscript was uploaded to the preprint server BioRxiv in 2017 (10.1101/134502).

3GOLD: optimized Levenshtein distance for clustering third-generation sequencing data.

BACKGROUND: Third-generation sequencing offers some advantages over next-generation sequencing predecessors, but with the caveat of harboring a much higher error rate. Clustering-related sequences is an essential task in modern biology. To accurately cluster sequences rich in errors, error type and frequency need to be accounted for. Levenshtein distance is a well-established mathematical algorithm for measuring the edit distance between words and can specifically weight insertions, deletions and substitutions. However, there are drawbacks to using Levenshtein distance in a biological context and hence has rarely been used for this purpose. We present novel modifications to the Levenshtein distance algorithm to optimize it for clustering error-rich biological sequencing data. RESULTS: We successfully introduced a bidirectional frameshift allowance with end-user determined accommodation caps combined with weighted error discrimination. Furthermore, our modifications dramatically improved the computational speed of Levenstein distance. For simulated ONT MinION and PacBio Sequel datasets, the average clustering sensitivity for 3GOLD was 41.45% (S.D. 10.39) higher than Sequence-Levenstein distance, 52.14% (S.D. 9.43) higher than Levenshtein distance, 55.93% (S.D. 8.67) higher than Starcode, 42.68% (S.D. 8.09) higher than CD-HIT-EST and 61.49% (S.D. 7.81) higher than DNACLUST. For biological ONT MinION data, 3GOLD clustering sensitivity was 27.99% higher than Sequence-Levenstein distance, 52.76% higher than Levenshtein distance, 56.39% higher than Starcode, 48% higher than CD-HIT-EST and 70.4% higher than DNACLUST. CONCLUSION: Our modifications to Levenshtein distance have improved its speed and accuracy compared to the classic Levenshtein distance, Sequence-Levenshtein distance and other commonly used clustering approaches on simulated and biological third-generation sequenced datasets. Our clustering approach is appropriate for datasets of unknown cluster centroids, such as those generated with unique molecular identifiers as well as known centroids such as barcoded datasets. A strength of our approach is high accuracy in resolving small clusters and mitigating the number of singletons.

LUCS: a high-resolution nucleic acid sequencing tool for accurate long-read analysis of individual DNA molecules.

Nucleic acid sequence analyses are fundamental to all aspects of biological research, spanning aging, mitochondrial DNA (mtDNA) and cancer, as well as microbial and viral evolution. Over the past several years, significant improvements in DNA sequencing, including consensus sequence analysis, have proven invaluable for high-throughput studies. However, all current DNA sequencing platforms have limited utility for studies of complex mixtures or of individual long molecules, the latter of which is crucial to understanding evolution and consequences of single nucleotide variants and their combinations. Here we report a new technology termed LUCS (Long-molecule UMI-driven Consensus Sequencing), in which reads from third-generation sequencing are aggregated by unique molecular identifiers (UMIs) specific for each individual DNA molecule. This enables in-silico reconstruction of highly accurate consensus reads of each DNA molecule independent of other molecules in the sample. Additionally, use of two UMIs enables detection of artificial recombinants (chimeras). As proof of concept, we show that application of LUCS to assessment of mitochondrial genomes in complex mixtures from single cells was associated with an error rate of 1X10-4 errors/nucleotide. Thus, LUCS represents a major step forward in DNA sequencing that offers high-throughput capacity and high-accuracy reads in studies of long DNA templates and nucleotide variants in heterogenous samples.

A nanoscale, multi-parametric flow cytometry-based platform to study mitochondrial heterogeneity and mitochondrial DNA dynamics.

Mitochondria are well-characterized regarding their function in both energy production and regulation of cell death; however, the heterogeneity that exists within mitochondrial populations is poorly understood. Typically analyzed as pooled samples comprised of millions of individual mitochondria, there is little information regarding potentially different functionality across subpopulations of mitochondria. Herein we present a new methodology to analyze mitochondria as individual components of a complex and heterogeneous network, using a nanoscale and multi-parametric flow cytometry-based platform. We validate the platform using multiple downstream assays, including electron microscopy, ATP generation, quantitative mass-spectrometry proteomic profiling, and mtDNA analysis at the level of single organelles. These strategies allow robust analysis and isolation of mitochondrial subpopulations to more broadly elucidate the underlying complexities of mitochondria as these organelles function collectively within a cell.

Impact of exercise on oocyte quality in the POLG mitochondrial DNA mutator mouse.

The mtDNA 'mutator' mouse, also called the 'POLG' mouse, is a well-characterized model frequently used for studies of progeroid aging. Harboring a mutation in the proofreading domain of the mitochondrial polymerase, polymerase-γ (Polg), POLG mice acquire mtDNA mutations at an accelerated rate. This results in premature mitochondrial dysfunction and a systemic aging phenotype. Previous work has demonstrated that the progeroid phenotype in POLG is attenuated following endurance exercise, the only reported intervention to extend health span and lifespan of these mice. Herein, oocyte quality was evaluated in sedentary and exercised POLG mice. In mice homozygous for the Polg mutation, litter size is dramatically reduced as compared to heterozygous Polg mice. Following ovarian hyper-stimulation, oocytes were retrieved until 9 months of age in exercised and sedentary groups, with no oocytes ovulated thereafter. Although ovulated oocyte numbers were not impacted by exercise, we did find a modest improvement in both the ovarian follicle reserve and in oocyte quality based on meiotic spindle assembly, chromosomal segregation and mitochondrial distribution at 7 months of age in exercised POLG mice as compared to sedentary counterparts. Of note, analysis of mtDNA mutational load revealed no differences between exercised and sedentary groups. Collectively, these data indicate that exercise differentially influences somatic tissues of the POLG mouse as compared to oocytes, highlighting important mechanistic differences between mitochondrial regulatory mechanisms in the soma and the germline.

Data on the time of integration of the human mitochondrial pseudogenes (NUMTs) into the nuclear genome.

The data and methods presented in this article are supplementing the research article "Integration of mtDNA pseudogenes into the nuclear genome coincides with speciation of the human genus. A hypothesis", DOI: 10.1016/j.mito.2016.12.001 (Gunbin et al., 2017) [1]. Mitochondrial DNA is known to get inserted into nuclear DNA to form NUMTs, i.e. nuclear DNA pseudogenes of the mtDNA. We present here the sequences of selected NUMTs, in which time of integration can be determined with sufficient precision. We report their chromosomal positions , their position within the great ape mtDNA phylogeny, and their times of integration into the nuclear genome. The methods used to generate the data and to control their quality are also presented. The dataset is made publicly available to enable critical or extended analyzes.

Integration of mtDNA pseudogenes into the nuclear genome coincides with speciation of the human genus. A hypothesis.

Fragments of mitochondrial DNA are known to get inserted into nuclear DNA to form NUMTs, i.e. nuclear pseudogenes of the mtDNA. The insertion of a NUMT is a rare event. Hundreds of pseudogenes have been cataloged in the human genome. NUMTs are, in essence, a special type of mutation with their own internal timer, which is synchronized with an established molecular clock, the mtDNA. Thus insertion of NUMTs can be timed with respect to evolution milestones such as the emergence of new species. We asked whether NUMTs were inserted uniformly over time or preferentially during certain periods of evolution, as implied by the "punctuated evolution" model. To our surprise, the NUMT insertion times do appear nonrandom with at least one cluster positioned at around 2.8 million years ago (Ma). Interestingly, 2.8Ma closely corresponds to the time of emergence of the genus Homo, and to a well-documented period of major climate change ca. 2.9-2.5Ma. It is tempting to hypothesize that the insertion of NUMTs is related to the speciation process. NUMTs could be either "riders", i.e., their insertion could be facilitated by the overall higher genome rearrangement activity during speciation, or "drivers", i.e. they may more readily get fixed in the population due to positive selection associated with speciation. If correct, the hypothesis would support the idea that evolution of our genus may have happened in a rapid, punctuated manner.

Amelioration of premature aging in mtDNA mutator mouse by exercise: the interplay of oxidative stress, PGC-1α, p53, and DNA damage. A hypothesis.

The mtDNA mutator mouse lacks the proofreading capacity of the sole mtDNA polymerase, leading to accumulation of somatic mtDNA mutations, and a profound premature aging phenotype including elevated oxidative stress and apoptosis, and reduced mitochondrial function. We have previously reported that endurance exercise alleviates the aging phenotype in the mutator mice, reduces oxidative stress, and enhances mitochondrial biogenesis. Here we summarize our findings, with the emphasis on the central role of p53 in these adaptations. We demonstrate that mtDNA in sedentary and exercised PolG mice carry similar amounts of mutations in muscle, but in addition to that sedentary mice have more non-mutational damage, which is mitigated by exercise. It follows therefore that the profound alleviation of the mtDNA mutator phenotype in muscle by exercise may not require a reduction in mtDNA mutational load, but rather a decrease of mtDNA damage and/or oxidative stress. We further hypothesize that the observed 'alleviation without a reduction of mutational load' implies that the oxidative stress in PolG muscle is maintained, at least in part, by the 'malicious cycle', a hypothetical positive feedback potentially driven by the 'transcriptional mutagenesis', that is the conversion of chemically modified nucleotides into mutant RNA bases by the mitochondrial RNA polymerase.

A YAP/TAZ-miR-130/301 molecular circuit exerts systems-level control of fibrosis in a network of human diseases and physiologic conditions.

The molecular origins of fibrosis affecting multiple tissue beds remain incompletely defined. Previously, we delineated the critical role of the control of extracellular matrix (ECM) stiffening by the mechanosensitive microRNA-130/301 family, as activated by the YAP/TAZ co-transcription factors, in promoting pulmonary hypertension (PH). We hypothesized that similar mechanisms may dictate fibrosis in other tissue beds beyond the pulmonary vasculature. Employing an in silico combination of microRNA target prediction, transcriptomic analysis of 137 human diseases and physiologic states, and advanced gene network modeling, we predicted the microRNA-130/301 family as a master regulator of fibrotic pathways across a cohort of seemingly disparate diseases and conditions. In two such diseases (pulmonary fibrosis and liver fibrosis), inhibition of microRNA-130/301 prevented the induction of ECM modification, YAP/TAZ, and downstream tissue fibrosis. Thus, mechanical forces act through a central feedback circuit between microRNA-130/301 and YAP/TAZ to sustain a common fibrotic phenotype across a network of human physiologic and pathophysiologic states. Such re-conceptualization of interconnections based on shared systems of disease and non-disease gene networks may have broad implications for future convergent diagnostic and therapeutic strategies.

Matrix Remodeling Promotes Pulmonary Hypertension through Feedback Mechanoactivation of the YAP/TAZ-miR-130/301 Circuit.

Pulmonary hypertension (PH) is a deadly vascular disease with enigmatic molecular origins. We found that vascular extracellular matrix (ECM) remodeling and stiffening are early and pervasive processes that promote PH. In multiple pulmonary vascular cell types, such ECM stiffening induced the microRNA-130/301 family via activation of the co-transcription factors YAP and TAZ. MicroRNA-130/301 controlled a PPAR?-APOE-LRP8 axis, promoting collagen deposition and LOX-dependent remodeling and further upregulating YAP/TAZ via a mechanoactive feedback loop. In turn, ECM remodeling controlled pulmonary vascular cell crosstalk via such mechanotransduction, modulation of secreted vasoactive effectors, and regulation of associated microRNA pathways. In vivo, pharmacologic inhibition of microRNA-130/301, APOE, or LOX activity ameliorated ECM remodeling and PH. Thus, ECM remodeling, as controlled by the YAP/TAZ-miR-130/301 feedback circuit, is an early PH trigger and offers combinatorial therapeutic targets for this devastating disease.

Genetic and hypoxic alterations of the microRNA-210-ISCU1/2 axis promote iron-sulfur deficiency and pulmonary hypertension.

Iron-sulfur (Fe-S) clusters are essential for mitochondrial metabolism, but their regulation in pulmonary hypertension (PH) remains enigmatic. We demonstrate that alterations of the miR-210-ISCU1/2 axis cause Fe-S deficiencies in vivo and promote PH. In pulmonary vascular cells and particularly endothelium, hypoxic induction of miR-210 and repression of the miR-210 targets ISCU1/2 down-regulated Fe-S levels. In mouse and human vascular and endothelial tissue affected by PH, miR-210 was elevated accompanied by decreased ISCU1/2 and Fe-S integrity. In mice, miR-210 repressed ISCU1/2 and promoted PH. Mice deficient in miR-210, via genetic/pharmacologic means or via an endothelial-specific manner, displayed increased ISCU1/2 and were resistant to Fe-S-dependent pathophenotypes and PH. Similar to hypoxia or miR-210 overexpression, ISCU1/2 knockdown also promoted PH. Finally, cardiopulmonary exercise testing of a woman with homozygous ISCU mutations revealed exercise-induced pulmonary vascular dysfunction. Thus, driven by acquired (hypoxia) or genetic causes, the miR-210-ISCU1/2 regulatory axis is a pathogenic lynchpin causing Fe-S deficiency and PH. These findings carry broad translational implications for defining the metabolic origins of PH and potentially other metabolic diseases sharing similar underpinnings.

The microRNA-130/301 family controls vasoconstriction in pulmonary hypertension.

Pulmonary hypertension (PH) is a complex disorder, spanning several known vascular cell types. Recently, we identified the microRNA-130/301 (miR-130/301) family as a regulator of multiple pro-proliferative pathways in PH, but the true breadth of influence of the miR-130/301 family across cell types in PH may be even more extensive. Here, we employed targeted network theory to identify additional pathogenic pathways regulated by miR-130/301, including those involving vasomotor tone. Guided by these predictions, we demonstrated, via gain- and loss-of-function experimentation in vitro and in vivo, that miR-130/301-specific control of the peroxisome proliferator-activated receptor γ regulates a panel of vasoactive factors communicating between diseased pulmonary vascular endothelial and smooth muscle cells. Of these, the vasoconstrictive factor endothelin-1 serves as an integral point of communication between the miR-130/301-peroxisome proliferator-activated receptor γ axis in endothelial cells and contractile function in smooth muscle cells. Thus, resulting from an in silico analysis of the architecture of the PH disease gene network coupled with molecular experimentation in vivo, these findings clarify the expanded role of the miR-130/301 family in the global regulation of PH. They further emphasize the importance of molecular cross-talk among the diverse cellular populations involved in PH.

An Argonaute 2 switch regulates circulating miR-210 to coordinate hypoxic adaptation across cells.

Complex organisms may coordinate molecular responses to hypoxia by specialized avenues of communication across multiple tissues, but these mechanisms are poorly understood. Plasma-based, extracellular microRNAs have been described, yet their regulation and biological functions in hypoxia remain enigmatic. We found a unique pattern of release of the hypoxia-inducible microRNA-210 (miR-210) from hypoxic and reoxygenated cells. This microRNA is also elevated in human plasma in physiologic and pathologic conditions of altered oxygen demand and delivery. Released miR-210 can be delivered to recipient cells, and the suppression of its direct target ISCU and mitochondrial metabolism is primarily evident in hypoxia. To regulate these hypoxia-specific actions, prolyl-hydroxylation of Argonaute 2 acts as a molecular switch that reciprocally modulates miR-210 release and intracellular activity in source cells as well as regulates intracellular activity in recipient cells after miR-210 delivery. Therefore, Argonaute 2-dependent control of released miR-210 represents a unique communication system that integrates the hypoxic response across anatomically distinct cells, preventing unnecessary activity of delivered miR-210 in normoxia while still preparing recipient tissues for incipient hypoxic stress and accelerating adaptation.

Systems-level regulation of microRNA networks by miR-130/301 promotes pulmonary hypertension.

Development of the vascular disease pulmonary hypertension (PH) involves disparate molecular pathways that span multiple cell types. MicroRNAs (miRNAs) may coordinately regulate PH progression, but the integrative functions of miRNAs in this process have been challenging to define with conventional approaches. Here, analysis of the molecular network architecture specific to PH predicted that the miR-130/301 family is a master regulator of cellular proliferation in PH via regulation of subordinate miRNA pathways with unexpected connections to one another. In validation of this model, diseased pulmonary vessels and plasma from mammalian models and human PH subjects exhibited upregulation of miR-130/301 expression. Evaluation of pulmonary arterial endothelial cells and smooth muscle cells revealed that miR-130/301 targeted PPARγ with distinct consequences. In endothelial cells, miR-130/301 modulated apelin-miR-424/503-FGF2 signaling, while in smooth muscle cells, miR-130/301 modulated STAT3-miR-204 signaling to promote PH-associated phenotypes. In murine models, induction of miR-130/301 promoted pathogenic PH-associated effects, while miR-130/301 inhibition prevented PH pathogenesis. Together, these results provide insight into the systems-level regulation of miRNA-disease gene networks in PH with broad implications for miRNA-based therapeutics in this disease. Furthermore, these findings provide critical validation for the evolving application of network theory to the discovery of the miRNA-based origins of PH and other diseases.

Bone morphogenetic protein-2 decreases microRNA-30b and microRNA-30c to promote vascular smooth muscle cell calcification.

BACKGROUND: Vascular calcification resembles bone formation and involves vascular smooth muscle cell (SMC) transition to an osteoblast-like phenotype to express Runx2, a master osteoblast transcription factor. One possible mechanism by which Runx2 protein expression is induced is downregulation of inhibitory microRNAs (miR). METHODS AND RESULTS: Human coronary artery SMCs (CASMCs) treated with bone morphogenetic protein-2 (BMP-2; 100 ng/mL) demonstrated a 1.7-fold (P<0.02) increase in Runx2 protein expression at 24 hours. A miR microarray and target prediction database analysis independently identified miR-30b and miR-30c (miR-30b-c) as miRs that regulate Runx2 expression. Real-time-polymerase chain reaction confirmed that BMP-2 decreased miR-30b and miR-30c expression. A luciferase reporter assay verified that both miR-30b and miR-30c bind to the 3'-untranslated region of Runx2 mRNA to regulate its expression. CASMCs transfected with antagomirs to downregulate miR-30b-c demonstrated significantly increased Runx2, intracellular calcium deposition, and mineralization. Conversely, forced expression of miR-30b-c by transfection with pre-miR-30b-c prevented the increase in Runx2 expression and mineralization of SMCs. Calcified human coronary arteries demonstrated higher levels of BMP-2 and lower levels of miR-30b than did noncalcified donor coronary arteries. CONCLUSIONS: BMP-2 downregulates miR-30b and miR-30c to increase Runx2 expression in CASMCs and promote mineralization. Strategies that modulate expression of miR-30b and miR-30c may influence vascular calcification.