Mitochondria Are Fundamental for the Emergence of Metazoans: On Metabolism, Genomic Regulation, and the Birth of Complex Organisms.

Out of many intracellular bacteria, only the mitochondria and chloroplasts abandoned their independence billions of years ago and became endosymbionts within the host eukaryotic cell. Consequently, one cannot grow eukaryotic cells without their mitochondria, and the mitochondria cannot divide outside of the cell, thus reflecting interdependence. Here, we argue that such interdependence underlies the fundamental role of mitochondrial activities in the emergence of metazoans. Several lines of evidence support our hypothesis: (a) Differentiation and embryogenesis rely on mitochondrial function; (b) mitochondrial metabolites are primary precursors for epigenetic modifications (such as methyl and acetyl), which are critical for chromatin remodeling and gene expression, particularly during differentiation and embryogenesis; and (c) mitonuclear coregulation adapted to accommodate both housekeeping and tissue-dependent metabolic needs. We discuss the evolution of the unique mitochondrial genetic system, mitochondrial metabolites, mitonuclear coregulation, and their critical roles in the emergence of metazoans and in human disorders.

Mutant C. elegans mitofusin leads to selective removal of mtDNA heteroplasmic deletions across generations to maintain fitness.

BACKGROUND: Mitochondrial DNA (mtDNA) is present at high copy numbers in animal cells, and though characterized by a single haplotype in each individual due to maternal germline inheritance, deleterious mutations and intact mtDNA molecules frequently co-exist (heteroplasmy). A number of factors, such as replicative segregation, mitochondrial bottlenecks, and selection, may modulate the exitance of heteroplasmic mutations. Since such mutations may have pathological consequences, they likely survive and are inherited due to functional complementation via the intracellular mitochondrial network. Here, we hypothesized that compromised mitochondrial fusion would hamper such complementation, thereby affecting heteroplasmy inheritance. RESULTS: We assessed heteroplasmy levels in three Caenorhabditis elegans strains carrying different heteroplasmic mtDNA deletions (ΔmtDNA) in the background of mutant mitofusin (fzo-1). Animals displayed severe embryonic lethality and developmental delay. Strikingly, observed phenotypes were relieved during subsequent generations in association with complete loss of ΔmtDNA molecules. Moreover, deletion loss rates were negatively correlated with the size of mtDNA deletions, suggesting that mitochondrial fusion is essential and sensitive to the nature of the heteroplasmic mtDNA mutations. Introducing the ΔmtDNA into a fzo-1;pdr-1;+/ΔmtDNA (PARKIN ortholog) double mutant resulted in a skewed Mendelian progeny distribution, in contrast to the normal distribution in the fzo-1;+/ΔmtDNA mutant, and severely reduced brood size. Notably, the ΔmtDNA was lost across generations in association with improved phenotypes. CONCLUSIONS: Taken together, our findings show that when mitochondrial fusion is compromised, deleterious heteroplasmic mutations cannot evade natural selection while inherited through generations. Moreover, our findings underline the importance of cross-talk between mitochondrial fusion and mitophagy in modulating the inheritance of mtDNA heteroplasmy.

Mitochondrial DNA Transcription and Its Regulation: An Evolutionary Perspective.

The bacterial heritage of mitochondria, as well as its independent genome [mitochondrial DNA (mtDNA)] and polycistronic transcripts, led to the view that mitochondrial transcriptional regulation relies on an evolutionarily conserved, prokaryotic-like system that is separated from the rest of the cell. Indeed, mtDNA transcription was previously thought to be governed by a few dedicated direct regulators, namely, the mitochondrial RNA polymerase (POLRMT), two transcription factors (TFAM and TF2BM), one transcription elongation (TEFM), and one known transcription termination factor (mTERF1). Recent findings have, however, revealed that known nuclear gene expression regulators are also involved in mtDNA transcription and have identified novel transcriptional features consistent with adaptation of the mitochondria to the regulatory environment of the precursor of the eukaryotic cell. Finally, whereas mammals follow the human mtDNA transcription pattern, other organisms notably diverge in terms of mtDNA transcriptional regulation. Hence, mtDNA transcriptional regulation is likely more evolutionary diverse than once thought.

Human primitive brain displays negative mitochondrial-nuclear expression correlation of respiratory genes.

Oxidative phosphorylation (OXPHOS), a fundamental energy source in all human tissues, requires interactions between mitochondrial (mtDNA)- and nuclear (nDNA)-encoded protein subunits. Although such interactions are fundamental to OXPHOS, bi-genomic coregulation is poorly understood. To address this question, we analyzed ∼8500 RNA-seq experiments from 48 human body sites. Despite well-known variation in mitochondrial activity, quantity, and morphology, we found overall positive mtDNA-nDNA OXPHOS genes' co-expression across human tissues. Nevertheless, negative mtDNA-nDNA gene expression correlation was identified in the hypothalamus, basal ganglia, and amygdala (subcortical brain regions, collectively termed the "primitive" brain). Single-cell RNA-seq analysis of mouse and human brains revealed that this phenomenon is evolutionarily conserved, and both are influenced by brain cell types (involving excitatory/inhibitory neurons and nonneuronal cells) and by their spatial brain location. As the "primitive" brain is highly oxidative, we hypothesized that such negative mtDNA-nDNA co-expression likely controls for the high mtDNA transcript levels, which enforce tight OXPHOS regulation, rather than rewiring toward glycolysis. Accordingly, we found "primitive" brain-specific up-regulation of lactate dehydrogenase B (LDHB), which associates with high OXPHOS activity, at the expense of LDHA, which promotes glycolysis. Analyses of co-expression, DNase-seq, and ChIP-seq experiments revealed candidate RNA-binding proteins and CEBPB as the best regulatory candidates to explain these phenomena. Finally, cross-tissue expression analysis unearthed tissue-dependent splice variants and OXPHOS subunit paralogs and allowed revising the list of canonical OXPHOS transcripts. Taken together, our analysis provides a comprehensive view of mito-nuclear gene co-expression across human tissues and provides overall insights into the bi-genomic regulation of mitochondrial activities.

A common pattern of DNase I footprinting throughout the human mtDNA unveils clues for a chromatin-like organization.

Human mitochondrial DNA (mtDNA) is believed to lack chromatin and histones. Instead, it is coated solely by the transcription factor TFAM. We asked whether mtDNA packaging is more regulated than once thought. To address this, we analyzed DNase-seq experiments in 324 human cell types and found, for the first time, a pattern of 29 mtDNA Genomic footprinting (mt-DGF) sites shared by ∼90% of the samples. Their syntenic conservation in mouse DNase-seq experiments reflect selective constraints. Colocalization with known mtDNA regulatory elements, with G-quadruplex structures, in TFAM-poor sites (in HeLa cells) and with transcription pausing sites, suggest a functional regulatory role for such mt-DGFs. Altered mt-DGF pattern in interleukin 3-treated CD34+ cells, certain tissue differences, and significant prevalence change in fetal versus nonfetal samples, offer first clues to their physiological importance. Taken together, human mtDNA has a conserved protein-DNA organization, which is likely involved in mtDNA regulation.

Initiation of mtDNA transcription is followed by pausing, and diverges across human cell types and during evolution.

Mitochondrial DNA (mtDNA) genes are long known to be cotranscribed in polycistrones, yet it remains impossible to study nascent mtDNA transcripts quantitatively in vivo using existing tools. To this end, we used deep sequencing (GRO-seq and PRO-seq) and analyzed nascent mtDNA-encoded RNA transcripts in diverse human cell lines and metazoan organisms. Surprisingly, accurate detection of human mtDNA transcription initiation sites (TISs) in the heavy and light strands revealed a novel conserved transcription pausing site near the light-strand TIS. This pausing site correlated with the presence of a bacterial pausing sequence motif, with reduced SNP density, and with a DNase footprinting signal in all tested cells. Its location within conserved sequence block 3 (CSBIII), just upstream of the known transcription-replication transition point, suggests involvement in such transition. Analysis of nonhuman organisms enabled de novo mtDNA sequence assembly, as well as detection of previously unknown mtDNA TIS, pausing, and transcription termination sites with unprecedented accuracy. Whereas mammals (Pan troglodytes, Macaca mulatta, Rattus norvegicus, and Mus musculus) showed a human-like mtDNA transcription pattern, the invertebrate pattern (Drosophila melanogaster and Caenorhabditis elegans) profoundly diverged. Our approach paves the path toward in vivo, quantitative, reference sequence-free analysis of mtDNA transcription in all eukaryotes.

Correction: Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates.

[This corrects the article DOI: 10.1371/journal.pbio.1002557.].

Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates.

The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m1A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m1A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m1A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function.

Ancient Out-of-Africa Mitochondrial DNA Variants Associate with Distinct Mitochondrial Gene Expression Patterns.

Mitochondrial DNA (mtDNA) variants have been traditionally used as markers to trace ancient population migrations. Although experiments relying on model organisms and cytoplasmic hybrids, as well as disease association studies, have served to underline the functionality of certain mtDNA SNPs, only little is known of the regulatory impact of ancient mtDNA variants, especially in terms of gene expression. By analyzing RNA-seq data of 454 lymphoblast cell lines from the 1000 Genomes Project, we found that mtDNA variants defining the most common African genetic background, the L haplogroup, exhibit a distinct overall mtDNA gene expression pattern, which was independent of mtDNA copy numbers. Secondly, intra-population analysis revealed subtle, yet significant, expression differences in four tRNA genes. Strikingly, the more prominent African mtDNA gene expression pattern best correlated with the expression of nuclear DNA-encoded RNA-binding proteins, and with SNPs within the mitochondrial RNA-binding proteins PTCD1 and MRPS7. Our results thus support the concept of an ancient regulatory transition of mtDNA-encoded genes as humans left Africa to populate the rest of the world.

Mitochondrial DNA associations with East Asian metabolic syndrome.

Mitochondrial dysfunction has repeatedly been reported associated with type 2 diabetes mellitus (T2DM) and metabolic syndrome (MS), as have mitochondrial DNA (mtDNA) tRNA and duplication mutations and mtDNA haplogroup lineages. We identified 19 Taiwanese T2DM and MS pedigrees from Taiwan, with putative matrilineal transmission, one of which harbored the pathogenic mtDNA tRNALeu(UUR) nucleotide (nt) 3243A>G mutation on the N9a3 haplogroup background. We then recruited three independent Taiwanese cohorts, two from Taipei (N = 498, mean age 52 and N = 1002, mean age 44) and one from a non-urban environment (N = 501, mean age 57). All three cohorts were assessed for an array of metabolic parameters, their mtDNA haplogroups determined, and the haplogroups correlated with T2DM/MS phenotypes. Logistic regression analysis revealed that mtDNA haplogroups D5, F4, and N9a conferred T2DM protection, while haplogroups F4 and N9a were risk factors for hypertension (HTN), and F4 was a risk factor for obesity (OB). Additionally, the 5263C>T (ND2 A165V) variant commonly associated with F4 was associated with hypertension (HTN). Cybrids were prepared with macro-haplogroup N (defined by variants m.ND3 10398A (114T) and m.ATP6 8701A (59T)) haplogroups B4 and F1 mtDNAs and from macro-haplogroup M (variants m.ND3 10398G (114A) and m.ATP6 8701G (59A)) haplogroup M9 mtDNAs. Additionally, haplogroup B4 and F1 cybrids were prepared with and without the mtDNA variant in ND1 3394T>C (Y30H) reported to be associated with T2DM. Assay of mitochondria complex I in these cybrids revealed that macro-haplogroup N cybrids had lower activity than M cybrids, that haplogroup F cybrids had lower activity than B4 cybrids, and that the ND1 3394T>C (Y30H) variant reduced complex I on both the B4 and F1 background but with very different cumulative effects. These data support the hypothesis that functional mtDNA variants may contribute to the risk of developing T2DM and MS.

RNA-DNA differences in human mitochondria restore ancestral form of 16S ribosomal RNA.

RNA transcripts are generally identical to the underlying DNA sequences. Nevertheless, RNA-DNA differences (RDDs) were found in the nuclear human genome and in plants and animals but not in human mitochondria. Here, by deep sequencing of human mitochondrial DNA (mtDNA) and RNA, we identified three RDD sites at mtDNA positions 295 (C-to-U), 13710 (A-to-U, A-to-G), and 2617 (A-to-U, A-to-G). Position 2617, within the 16S rRNA, harbored the most prevalent RDDs (>30% A-to-U and ∼15% A-to-G of the reads in all tested samples). The 2617 RDDs appeared already at the precursor polycistrone mitochondrial transcript. By using traditional Sanger sequencing, we identified the A-to-U RDD in six different cell lines and representative primates (Gorilla gorilla, Pongo pigmaeus, and Macaca mulatta), suggesting conservation of the mechanism generating such RDD. Phylogenetic analysis of more than 1700 vertebrate mtDNA sequences supported a thymine as the primate ancestral allele at position 2617, suggesting that the 2617 RDD recapitulates the ancestral 16S rRNA. Modeling U or G (the RDDs) at position 2617 stabilized the large ribosomal subunit structure in contrast to destabilization by an A (the pre-RDDs). Hence, these mitochondrial RDDs are likely functional.

A genetic view of the mitochondrial role in ageing: killing us softly.

In contrast to the nuclear genome, the mitochondrial DNA (mtDNA) is maternally inherited and resides in multiple cellular copies that may vary in sequence (heteroplasmy). Although the interaction between mtDNA and nuclear DNA-encoded factors (mito-nuclear interaction) is vital, the mtDNA accumulates mutations an order of magnitude faster than the nuclear genome both during evolution and during the lifetime of the individual, thus requiring tight mito-nuclear co-evolution. These unique features drew the attention of many to suggest a role for the mitochondria in ageing. Although an excess of mtDNA mutations has been found in aged humans and animal models, most of these mutations had minor functional potential. Moreover, there are mtDNA mutations that recur in aged humans, but do not have any clear functionality. Nevertheless, accumulation of recurrent private mutations with minor functionality in the fast-ageing, mtDNA polymerase mutated mice (Pol-gamma), suggested that these very mtDNA alterations participate in ageing. This introduces a paradox: how would either single or recurrent mutations with negligible functionality play a role in a major chronic phenotype such as ageing?Here, we propose a hypothesis to partially resolve this paradox: accumulation of mitochondrial mutations with subtle functionality, which was overlooked by the mechanisms of selection, supplemented by slightly affected fusion-fission cycles, will hamper mitochondrial functional complementation within cells, disrupt mito-nuclear interactions and lead to ageing. Since certain mito-nuclear genotypes are less functionally compatible than others, and since the mtDNA and the nuclear genome segregate independently among generations, mild functionality of mutations will have differential effect on individuals in the population thus explaining the large variability in the ageing phenotype even within ethnic groups. We emphasize the role of recurrent mtDNA mutations with functional potential during evolution and during the lifetime of the individual.

Mitochondrial DNA heteroplasmy in diabetes and normal adults: role of acquired and inherited mutational patterns in twins.

Heteroplasmy, the mixture of mitochondrial genomes (mtDNA), varies among individuals and cells. Heteroplasmy levels alter the penetrance of pathological mtDNA mutations, and the susceptibility to age-related diseases such as Parkinson's disease. Although mitochondrial dysfunction occurs in age-related type 2 diabetes mellitus (T2DM), the involvement of heteroplasmy in diabetes is unclear. We hypothesized that the heteroplasmic mutational (HM) pattern may change in T2DM. To test this, we used next-generation sequencing, i.e. massive parallel sequencing (MPS), along with PCR-cloning-Sanger sequencing to analyze HM in blood and skeletal muscle DNA samples from monozygotic (MZ) twins either concordant or discordant for T2DM. Great variability was identified in the repertoires and amounts of HMs among individuals, with a tendency towards more mutations in skeletal muscle than in blood. Whereas many HMs were unique, many were either shared among twin pairs or among tissues of the same individual, regardless of their prevalence. This suggested a heritable influence on even low abundance HMs. We found no clear differences between T2DM and controls. However, we found ~5-fold increase of HMs in non-coding sequences implying the influence of negative selection (P < 0.001). This negative selection was evident both in moderate to highly abundant heteroplasmy (>5% of the molecules per sample) and in low abundance heteroplasmy (<5% of the molecules). Although our study found no evidence supporting the involvement of HMs in the etiology of T2DM, the twin study found clear evidence of a heritable influence on the accumulation of HMs as well as the signatures of selection in heteroplasmic mutations.

Mitochondrial-nuclear co-evolution and its effects on OXPHOS activity and regulation.

Factors required for mitochondrial function are encoded both by the nuclear and mitochondrial genomes. The order of magnitude higher mutation rate of animal mitochondrial DNA (mtDNA) enforces tight co-evolution of mtDNA and nuclear DNA encoded factors. In this essay we argue that such co evolution exists at the population and inter-specific levels and affect disease susceptibility. We also argue for the existence of three modes of co-evolution in the mitochondrial genetic system, which include the interaction of mtDNA and nuclear DNA encoded proteins, nuclear protein - mtDNA-encoded RNA interaction within the mitochondrial translation machinery and nuclear DNA encoded proteins-mtDNA binging sites interaction in the frame of the mtDNA replication and transcription machineries. These modes of co evolution require co-regulation of the interacting factors encoded by the two genomes. Thus co evolution plays an important role in modulating mitochondrial activity. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

CHILD: a new tool for detecting low-abundance insertions and deletions in standard sequence traces.

Several methods have been proposed for detecting insertion/deletions (indels) from chromatograms generated by Sanger sequencing. However, most such methods are unsuitable when the mutated and normal variants occur at unequal ratios, such as is expected to be the case in cancer, with organellar DNA or with alternatively spliced RNAs. In addition, the current methods do not provide robust estimates of the statistical confidence of their results, and the sensitivity of this approach has not been rigorously evaluated. Here, we present CHILD, a tool specifically designed for indel detection in mixtures where one variant is rare. CHILD makes use of standard sequence alignment statistics to evaluate the significance of the results. The sensitivity of CHILD was tested by sequencing controlled mixtures of deleted and undeleted plasmids at various ratios. Our results indicate that CHILD can identify deleted molecules present as just 5% of the mixture. Notably, the results were plasmid/primer-specific; for some primers and/or plasmids, the deleted molecule was only detected when it comprised 10% or more of the mixture. The false positive rate was estimated to be lower than 0.4%. CHILD was implemented as a user-oriented web site, providing a sensitive and experimentally validated method for the detection of rare indel-carrying molecules in common Sanger sequence reads.

The metazoan landscape of mitochondrial DNA gene order and content is shaped by selection and affects mitochondrial transcription.

Mitochondrial DNA (mtDNA) harbors essential genes in most metazoans, yet the regulatory impact of the multiple evolutionary mtDNA rearrangements has been overlooked. Here, by analyzing mtDNAs from ~8000 metazoans we found high gene content conservation (especially of protein and rRNA genes), and codon preferences for mtDNA-encoded tRNAs across most metazoans. In contrast, mtDNA gene order (MGO) was selectively constrained within but not between phyla, yet certain gene stretches (ATP8-ATP6, ND4-ND4L) were highly conserved across metazoans. Since certain metazoans with different MGOs diverge in mtDNA transcription, we hypothesized that evolutionary mtDNA rearrangements affected mtDNA transcriptional patterns. As a first step to test this hypothesis, we analyzed available RNA-seq data from 53 metazoans. Since polycistron mtDNA transcripts constitute a small fraction of the steady-state RNA, we enriched for polycistronic boundaries by calculating RNA-seq read densities across junctions between gene couples encoded either by the same strand (SSJ) or by different strands (DSJ). We found that organisms whose mtDNA is organized in alternating reverse-strand/forward-strand gene blocks (mostly arthropods), displayed significantly reduced DSJ read counts, in contrast to organisms whose mtDNA genes are preferentially encoded by one strand (all chordates). Our findings suggest that mtDNA rearrangements are selectively constrained and likely impact mtDNA regulation.

Ancient mtDNA genetic variants modulate mtDNA transcription and replication.

Although the functional consequences of mitochondrial DNA (mtDNA) genetic backgrounds (haplotypes, haplogroups) have been demonstrated by both disease association studies and cell culture experiments, it is not clear which of the mutations within the haplogroup carry functional implications and which are "evolutionary silent hitchhikers". We set forth to study the functionality of haplogroup-defining mutations within the mtDNA transcription/replication regulatory region by in vitro transcription, hypothesizing that haplogroup-defining mutations occurring within regulatory motifs of mtDNA could affect these processes. We thus screened >2500 complete human mtDNAs representing all major populations worldwide for natural variation in experimentally established protein binding sites and regulatory regions comprising a total of 241 bp in each mtDNA. Our screen revealed 77/241 sites showing point mutations that could be divided into non-fixed (57/77, 74%) and haplogroup/sub-haplogroup-defining changes (i.e., population fixed changes, 20/77, 26%). The variant defining Caucasian haplogroup J (C295T) increased the binding of TFAM (Electro Mobility Shift Assay) and the capacity of in vitro L-strand transcription, especially of a shorter transcript that maps immediately upstream of conserved sequence block 1 (CSB1), a region associated with RNA priming of mtDNA replication. Consistent with this finding, cybrids (i.e., cells sharing the same nuclear genetic background but differing in their mtDNA backgrounds) harboring haplogroup J mtDNA had a >2 fold increase in mtDNA copy number, as compared to cybrids containing haplogroup H, with no apparent differences in steady state levels of mtDNA-encoded transcripts. Hence, a haplogroup J regulatory region mutation affects mtDNA replication or stability, which may partially account for the phenotypic impact of this haplogroup. Our analysis thus demonstrates, for the first time, the functional impact of particular mtDNA haplogroup-defining control region mutations, paving the path towards assessing the functionality of both fixed and un-fixed genetic variants in the mitochondrial genome.