MIWI2, a P element-induced wimpy testes (PIWI) argonaute protein known for suppressing retrotransposons during male gonadogenesis, has an unexplored role in mammalian somatic cells. We identify MIWI2 multiciliated (M2MC) cells as a rare subset of airway multiciliated cells and investigate MIWI2's function in antiviral host defense. We analyzed transcriptomes from Miwi2 heterozygous (Miwi2 +/tom) and deficient (Miwi2 tom/tom) mice following influenza A infection. During infection, Miwi2 deficiency was associated with reduced mitochondrial and ribosomal gene expression in M2MC cells, increased mitochondrial reactive oxygen species (ROS) production and ADP/ATP ratios in multiciliated cells, and enhanced viral clearance and recovery. Additionally, Miwi2-expressing cells exhibited reduced levels of small RNAs derived from nuclear mitochondrial DNA. These findings reveal a previously unrecognized role for Miwi2 in regulating small non-coding RNAs and mitochondrial oxidant production in somatic cells, indicating a function beyond its established germline activities. Our study identifies Miwi2/Piwil4 as a potential factor influencing susceptibility to severe respiratory infections.

This study evaluates the efficacy of rapid clinical exome sequencing (CES) and mitochondrial DNA (mtDNA) sequencing for diagnosing genetic disorders in critically ill pediatric patients.

A multi-centre investigation was conducted, enrolling critically ill pediatric patients suspected of having genetic disorders from March 2019 to December 2020. Peripheral blood samples from patients and their parents were analyzed using CES (proband-parent) and mtDNA sequencing (proband-mother) based on Next-Generation Sequencing (NGS) technology.

The study included 44 pediatric patients (24 males, 20 females) with a median age of 27 days. The median turnaround time for genetic tests was 9.5 days. Genetic disorders were diagnosed in 25 patients (56.8%): 5 with chromosome microduplication/deletion syndromes (11.3%), 1 with UPD-related disease (2.3%), and 19 with monogenic diseases (43.2%). De novo variants were identified in nine patients (36.0%). A neonate was diagnosed with two genetic disorders due to a homozygous SLC25A20 variant and an MT-TL1 gene variation.

Rapid genetic diagnosis is crucial for critically ill pediatric patients with suspected genetic disorders. CES and mtDNA sequencing offer precise and timely results, guiding treatment and reducing mortality and disability, making them suitable primary diagnostic tools.

The personal identification of monozygotic (MZ) twins is of great importance in forensic medicine. Due to the extreme similarity in genetic between MZ twins, it is challenging to differentiate them using autosomal STR genotyping. Forensic experts are striving to explore available genetic markers that can differentiate between MZ twins. With the advent of next-generation sequence (NGS), an increasing number of genetic markers have been demonstrated to effectively differentiate between MZ twins. Here, we summarized for the relevant studies on MZ twins' differentiation and discussed the limitations of the underlying markers. In details, single-nucleotide variants (SNVs), copy number variation (CNV), mitochondrial DNA (mtDNA), DNA methylation, and non-coding RNA have been demonstrated considerable value. Furthermore, the utilization of proteomics, metabolomics, and microbiomics has shed light on MZ twin differentiation. Additionally, we introduce the methodologies for MZ differentiation based on external morphological variations observed in the human body. Looking to the future, the process of aging may represent a novel avenue for the differentiation of MZ twins.

The unique features of mitochondrial DNA (mtDNA), including its circular and multicopy nature, the possible coexistence of wild-type and mutant molecules (i.e., heteroplasmy) and the presence of nuclear mitochondrial DNA segments (NUMTs), make the diagnosis of mtDNA diseases particularly challenging. The extensive deployment of next-generation sequencing (NGS) technologies has significantly advanced the diagnosis of mtDNA-related diseases. However, the vast amounts and diverse types of sequencing data complicate the interpretation of these variants. From sequence alignment to variant calling, NGS-based mtDNA sequencing requires specialized bioinformatics tools, adapted for the mitochondrial genome. This study presents the use of new bioinformatics approaches, optimized for short- and long-read sequencing data, to enhance the accuracy of mtDNA analysis in diagnostics. Two recent and emerging free bioinformatics tools, Mitopore and MitoSAlt, were evaluated on patients previously diagnosed with single nucleotide variants or large-scale deletions. Analyses were performed in Linux-based environments and web servers implemented in Python, Perl, Java, and R. The results indicated that each tool demonstrated high sensitivity and specific accuracy in identifying and quantifying various types of pathogenic variants. The study suggests that the integrated and parallel use of these tools offers a significant advantage over traditional methods in interpreting mtDNA genetic variants, reducing the computational demands, and provides an accurate diagnostic solution.

Nuclear mitochondrial DNA sequences (NUMTs) are mitochondrial DNA fragments in the nuclear genome, and their unclear distribution in Chrysomelidae species hinders the selection of accurate molecular markers for species identification and phylogenetic analysis. Our study presents a genome-wide survey of NUMTs in 32 Chrysomelidae species. Filtering strategies based on sequence length and open reading frame (ORF) features were employed to identify mitochondrial protein-coding genes (PCGs) minimally affected by NUMTs. Phylogenetic relationships were inferred from both mitochondrial PCG datasets and a COX1 dataset containing NUMTs. Our results show that NUMTs are chromosomally specific, species-specific, and widely distributed. ATP8, COX1, ND1, and ND4 are identified as relatively reliable molecular markers. Phylogenetic analysis is influenced by NUMTs and other factors such as sequence type and saturation. A total of 66 independent COX1 gene nuclear integration events were estimated across 32 species, mostly from distinct mitochondrial lineages. These findings suggest that NUMTs reflect key evolutionary processes such as gene flow and mitochondrial lineage diversification. Their prevalence emphasizes the need for refined molecular markers in species identification and phylogenetic analysis, while also highlighting the importance of NUMTs in understanding mitochondrial DNA integration and their contribution to species' evolutionary history.

Copepods of the genus Pennella parasitize a wide range of marine animals, including cetaceans, teleosts, and cephalopods worldwide. Their taxonomy is unclear, as there is incongruence between morphological and genetic data and incomplete species coverage. This study provides new morphological and genetic (COI) data from 23 specimens of Pennella cf. filosa (syn. P. balaenoptera) from western Mediterranean whales and a swordfish. First, their position in the phylogeny of Pennella was assessed and species delimitation revisited using all available Pennella COI sequences (n = 189), obtained from Mediterranean and north Pacific specimens from 18 host species (including multiple cetaceans and teleosts). Second, it was investigated whether the geographic location, degree of host vagility, or host taxonomic identity help explain genetic differentiation. Five distinct haplotype groups with varying genetic divergence were distinguished. Although the presence of sibling species cannot be ruled out, species delimitation methods could not find interspecific genetic differences, leaving the taxonomy of the genus unresolved. The observed genetic differentiation could not be attributed to geography or host type. This suggests that members of the genus Pennella show low specificity for definitive hosts and interoceanic dispersal mediated by some vagile definitive hosts. The use of more genetic markers for addressing these questions in the future is encouraged.

Mitochondrial DNA (mtDNA) is often more susceptible to damage compared to nuclear DNA. This is due to its localization in the mitochondrial matrix, where a large portion of reactive oxygen species are produced. Mitochondria do not have histones and mtDNA is only slightly protected by histone-like proteins and is believed to have less efficient repair mechanisms. In this review, we discuss the long-range PCR method, which allows for the effective detection of mtDNA damage. The method is based on the assumption that various types of DNA lesions can interfere the progress of DNA polymerase, resulting in reduced amplification efficiency. It can be used to estimate the number of additional (above background) lesions in mtDNA. The review outlines the evolution of the methodology, its variations, applications in a wide range of model organisms, the advantages of the method and its limitations, as well as ways to overcome these limitations. Over the past two decades, the use of long-range PCR has allowed the study of mtDNA repair mechanisms, the characteristics of mitochondrial genome damage in various neurodegenerative diseases, aging, ischemic and oncological processes, as well as in anticancer therapy. The assessment of mtDNA damage has also been proposed for use in environmental biomonitoring. This review provides a critical evaluation of the various variations of this method, summarizes the accumulated data, and discusses the role of mtDNA damage in different organs at the organismal level.

A recent study proposed a new genetic lineage of leatherback turtles (Dermochelys coriacea) based on genetic analysis, environmental history, and local ecological knowledge (LEK), suggesting the existence of two possible species or subspecies on the beaches of Oaxaca, diverging ~ 13.5 Mya. However, this hypothesis may be influenced by nuclear mitochondrial DNA segments (NUMTs), which could have been misamplified as true mtDNA. NUMTs are sequences that have migrated from the mitochondrial genome to the nuclear genome and can co-amplify with mtDNA, potentially leading to erroneous phylogenetic interpretations. We re-examined the evidence for this proposed lineage by reviewing taxonomic literature and additional genetic data. Our analysis indicates that the divergent sequences, previously associated with a new lineage of D. coriacea, are NUMTs rather than true mitochondrial sequences. This is the first evidence of NUMTs in sea turtles. We also proposed a more specific primer for the mitochondrial control region (D-loop) for leatherback turtles to avoid amplifying nuclear copies. Our findings highlight the importance of rigorous genetic validation in conservation genetics, where misinterpretations can significantly impact species management. Finally, we developed a general protocol for detecting NUMTs applicable to any species.

The presence of mitochondrial sequences in the nuclear genome (Numts) confounds analyses of mitochondrial sequence variation, and is a potential source of false positives in disease studies. To improve the analysis of mitochondrial variation in canines, we completed a systematic assessment of Numt content across genome assemblies, canine populations and the carnivore lineage.

Centering our analysis on the UU_Cfam_GSD_1.0/canFam4/Mischka assembly, a commonly used reference in dog genetic variation studies, we found a total of 321 Numts located throughout the nuclear genome and encompassing the entire sequence of the mitochondria. A comparison with 14 canine genome assemblies identified 63 Numts with presence-absence dimorphism among dogs, wolves, and a coyote. Furthermore, a subset of Numts were maintained across carnivore evolutionary time (arctic fox, polar bear, cat), with eight sequences likely more than 10 million years old, and shared with the domestic cat. On a population level, using structural variant data from the Dog10K Consortium for 1879 dogs and wolves, we identified 11 Numts that are absent in at least one sample, as well as 53 Numts that are absent from the Mischka assembly.

We highlight scenarios where the presence of Numts is a potentially confounding factor and provide an annotation of these sequences in canine genome assemblies. This resource will aid the identification and interpretation of polymorphisms in both somatic and germline mitochondrial studies in canines.

Multicopied mitogenome are prone to mutation during replication often resulting in heteroplasmy. The derived variants in a cell, organ, or an individual animal constitute a mitogene pool. The individual mitogene pool is initiated by a small fraction of the egg mitogene pool. However, the characteristics and relationship between them has not yet been investigated. This study quantitatively analyzed the heteroplasmy landscape, genetic loads, and selection strength of the mitogene pool of egg and hatchling in the silver carp (Hypophthalmichthys molitrix) using high-throughput resequencing. The results showed heteroplasmic sites distribute across the whole mitogenome in both eggs and hatchlings. The dominant substitution was Transversion in eggs and Transition in hatching accounting for 95.23%±2.07% and 85.38%±6.94% of total HP sites, respectively. The total genetic loads were 0.293±0.044 in eggs and 0.228±0.022 in hatchlings (P=0.048). The dN/dS ratio was 58.03±38.98 for eggs and 9.44±3.93 for hatchlings (P=0.037). These results suggest that the mitogenomes were under strong positive selection in eggs with tolerance to variants with deleterious effects, while the selection was positive but much weaker in hatchlings showing marked quality control. Based on these findings, we proposed a trans-generation dynamics model to explain differential development mode of the two mitogene pool between oocyte maturation and ontogenesis of offspring. This study sheds light on significance of mitogene pool for persistence of populations and subsequent integration in ecological studies and conservation practices.

The transition of detached fragments of mitochondrial DNA into the nucleus and their integration into chromosomal DNA is a special kind of genetic variability that highlights the relation between the two genomes and their interaction in a eukaryotic cell. The human genome contains several hundreds of insertions of mtDNA fragments (NUMTS). This paper presents an overview of the current state of research in this area. To date, evidence has been obtained that the occurrence of new mtDNA insertions in the nuclear genome is a seldom but not exceptionally rare event. The integration of new mtDNA fragments into the nuclear genome occurs during double-strand DNA break repair through the non-homologous end joining mechanism. Along with evolutionarily stable "genetic fossils" that were integrated into the nuclear genome millions of years ago and are shared by many species, there are NUMTS that could be species-specific, polymorphic in a species, or "private". Partial copies of mitochondrial DNA in the human nuclear genome can interfere with mtDNA during experimental studies of the mitochondrial genome, such as genotyping, heteroplasmy assessment, mtDNA methylation analysis, and mtDNA copy number estimation. In some cases, the insertion of multiple copies of the complete mitochondrial genome sequence may mimic paternal inheritance of mtDNA. The functional significance of NUMTS is poorly understood. For instance, they may be a source of variability for expression and splicing modulation. The role of NUMTS as a cause of hereditary diseases is negligible, since only a few cases of diseases caused by NUMTS have been described so far. In addition, NUMTS can serve as markers for evolutionary genetic studies. Of particular interest is the meaning of NUMTS in eukaryotic genome evolution. The constant flow of functionally inactive DNA sequences from mitochondria into the nucleus and its significance could be studied in view of the modern concepts of evolutionary theory suggesting non-adaptive complexity and the key role of stochastic processes in the formation of genomic structure.

Molluscan mitochondrial genomes are unusual because they show wide variation in size, radical genome rearrangements and frequently show high variation (> 10%) within species. As progress in understanding this variation has been limited, we used whole genome sequencing of a six-generation matriline of the terrestrial snail Cepaea nemoralis, as well as whole genome sequences from wild-collected C. nemoralis, the sister species C. hortensis, and multiple other snail species to explore the origins of mitochondrial DNA (mtDNA) variation. The main finding is that a high rate of SNP heteroplasmy in somatic tissue was negatively correlated with mtDNA copy number in both Cepaea species. In individuals with under ten mtDNA copies per nuclear genome, more than 10% of all positions were heteroplasmic, with evidence for transmission of this heteroplasmy through the germline. Further analyses showed evidence for purifying selection acting on non-synonymous mutations, even at low frequency of the rare allele, especially in cytochrome oxidase subunit 1 and cytochrome b. The mtDNA of some individuals of Cepaea nemoralis contained a length heteroplasmy, including up to 12 direct repeat copies of tRNA-Val, with 24 copies in another snail, Candidula rugosiuscula, and repeats of tRNA-Thr in C. hortensis. These repeats likely arise due to error prone replication but are not correlated with mitochondrial copy number in C. nemoralis. Overall, the findings provide key insights into mechanisms of replication, mutation and evolution in molluscan mtDNA, and so will inform wider studies on the biology and evolution of mtDNA across animal phyla.

Membrane contact sites (MCS) between mitochondria and the nucleus have been recently described. Termed nucleus associated mitochondria (NAM), they prime the expression of genes required for cellular resistance to stressors, thus offering a tethering mechanism for homeostatic communication. Here, we discuss the composition of NAM and their physiological and pathological significance.

Paeonia lactiflora (P. lactiflora), a perennial plant renowned for its medicinal roots, provides a unique case for studying the phylogenetic relationships of species based on organelle genomes, as well as the transference of DNA across organelle genomes. In order to investigate this matter, we sequenced and characterized the mitochondrial genome (mitogenome) of P. lactiflora. Similar to the chloroplast genome (cpgenome), the mitogenome of P. lactiflora extends across 181,688 base pairs (bp). Its unique quadripartite structure results from a pair of extensive inverted repeats, each measuring 25,680 bp in length. The annotated mitogenome includes 27 protein-coding genes, 37 tRNAs, 8 rRNAs, and two pseudogenes (rpl5, rpl16). Phylogenetic analysis was performed to identify phylogenetic trees consistent with Paeonia species phylogeny in the APG Ⅳ system. Moreover, a total of 12 MTPT events were identified and 32 RNA editing sites were detected during mitogenome analysis of P. lactiflora. Our research successfully compiled and annotated the mitogenome of P. lactiflora. The study provides valuable insights regarding the taxonomic classification and molecular evolution within the Paeoniaceae family.

Until a few decades ago, most of our knowledge of RNA transcription products was focused on protein-coding sequences, which were later determined to make up the smallest portion of the mammalian genome. Since 2002, we have learnt a great deal about the intriguing world of non-coding RNAs (ncRNAs), mainly due to the rapid development of bioinformatic tools and next-generation sequencing (NGS) platforms. Moreover, interest in non-human ncRNAs and their functions has increased as a result of these technologies and the accessibility of complete genome sequences of species ranging from Archaea to primates. Despite not producing proteins, ncRNAs constitute a vast family of RNA molecules that serve a number of regulatory roles and are essential for cellular physiology and pathology. This review focuses on a subgroup of human ncRNAs, namely mtDNA-encoded long non-coding RNAs (mt-lncRNAs), which are transcribed from the mitochondrial genome and whose disparate localisations and functions are linked as much to mitochondrial metabolism as to cellular physiology and pathology.

Eukaryotic life depends on the functional elements encoded by both the nuclear genome and organellar genomes, such as those contained within the mitochondria. The content, size, and structure of the mitochondrial genome varies across organisms with potentially large implications for phenotypic variance and resulting evolutionary trajectories. Among yeasts in the subphylum Saccharomycotina, extensive differences have been observed in various species relative to the model yeast Saccharomyces cerevisiae, but mitochondrial genome sampling across many groups has been scarce, even as hundreds of nuclear genomes have become available.

By extracting mitochondrial assemblies from existing short-read genome sequence datasets, we have greatly expanded both the number of available genomes and the coverage across sparsely sampled clades.

Comparison of 353 yeast mitochondrial genomes revealed that, while size and GC content were fairly consistent across species, those in the genera Metschnikowia and Saccharomyces trended larger, while several species in the order Saccharomycetales, which includes S. cerevisiae, exhibited lower GC content. Extreme examples for both size and GC content were scattered throughout the subphylum. All mitochondrial genomes shared a core set of protein-coding genes for Complexes III, IV, and V, but they varied in the presence or absence of mitochondrially-encoded canonical Complex I genes. We traced the loss of Complex I genes to a major event in the ancestor of the orders Saccharomycetales and Saccharomycodales, but we also observed several independent losses in the orders Phaffomycetales, Pichiales, and Dipodascales. In contrast to prior hypotheses based on smaller-scale datasets, comparison of evolutionary rates in protein-coding genes showed no bias towards elevated rates among aerobically fermenting (Crabtree/Warburg-positive) yeasts. Mitochondrial introns were widely distributed, but they were highly enriched in some groups. The majority of mitochondrial introns were poorly conserved within groups, but several were shared within groups, between groups, and even across taxonomic orders, which is consistent with horizontal gene transfer, likely involving homing endonucleases acting as selfish elements.

As the number of available fungal nuclear genomes continues to expand, the methods described here to retrieve mitochondrial genome sequences from these datasets will prove invaluable to ensuring that studies of fungal mitochondrial genomes keep pace with their nuclear counterparts.

Retrotransposons are highly prevalent in most animals and account for more than 35% of the human genome. However, the prevalence, biogenesis mechanism and function of retrotransposons remain largely unknown. Here, we developed retroSeeker, a novel computational software that identifies novel retrotransposons from pairwise alignments of genomes and decodes their biogenesis, expression, evolution and potential functions. We discovered that the majority of new retrotransposons exhibit a specific L1 endonuclease cleavage motif, with some motifs precisely located ten nucleotides upstream of the insertion site. We identified that a large number of candidate functional genes might be generated through a retrotransposition mechanism. Importantly, we uncovered previously uncharacterized classes of retrotransposons related to histone genes, mitochondrial genes and vault RNAs. Moreover, we elucidated the tissue-specific expression of retrotransposons and demonstrated their ubiquitous expression in various cancer types. We also revealed the complex evolutionary patterns of retrotransposons and identified numerous species-specific retrotransposition events. Taken together, our findings establish a paradigm for discovering novel classes of retrotransposons and elucidating their new characteristics in any species.

Eukaryotic life depends on the functional elements encoded by both the nuclear genome and organellar genomes, such as those contained within the mitochondria. The content, size, and structure of the mitochondrial genome varies across organisms with potentially large implications for phenotypic variance and resulting evolutionary trajectories. Among yeasts in the subphylum Saccharomycotina, extensive differences have been observed in various species relative to the model yeast Saccharomyces cerevisiae, but mitochondrial genome sampling across many groups has been scarce, even as hundreds of nuclear genomes have become available. By extracting mitochondrial assemblies from existing short-read genome sequence datasets, we have greatly expanded both the number of available genomes and the coverage across sparsely sampled clades. Comparison of 353 yeast mitochondrial genomes revealed that, while size and GC content were fairly consistent across species, those in the genera Metschnikowia and Saccharomyces trended larger, while several species in the order Saccharomycetales, which includes S. cerevisiae, exhibited lower GC content. Extreme examples for both size and GC content were scattered throughout the subphylum. All mitochondrial genomes shared a core set of protein-coding genes for Complexes III, IV, and V, but they varied in the presence or absence of mitochondrially-encoded canonical Complex I genes. We traced the loss of Complex I genes to a major event in the ancestor of the orders Saccharomycetales and Saccharomycodales, but we also observed several independent losses in the orders Phaffomycetales, Pichiales, and Dipodascales. In contrast to prior hypotheses based on smaller-scale datasets, comparison of evolutionary rates in protein-coding genes showed no bias towards elevated rates among aerobically fermenting (Crabtree/Warburg-positive) yeasts. Mitochondrial introns were widely distributed, but they were highly enriched in some groups. The majority of mitochondrial introns were poorly conserved within groups, but several were shared within groups, between groups, and even across taxonomic orders, which is consistent with horizontal gene transfer, likely involving homing endonucleases acting as selfish elements. As the number of available fungal nuclear genomes continues to expand, the methods described here to retrieve mitochondrial genome sequences from these datasets will prove invaluable to ensuring that studies of fungal mitochondrial genomes keep pace with their nuclear counterparts.