Liron Levin

Disrupting Mitochondrial–Nuclear Coevolution Affects OXPHOS Complex I Integrity and Impacts Human Health

The mutation rate of the mitochondrial DNA (mtDNA), which is higher by an order of magnitude as compared with the nuclear genome, enforces tight mitonuclear coevolution to maintain mitochondrial activities. Interruption of such coevolution plays a role in interpopulation hybrid breakdown, speciation events, and disease susceptibility. Previously, we found an elevated amino acid replacement rate and positive selection in the nuclear DNA-encoded oxidative phosphorylation (OXPHOS) complex I subunit NDUFC2, a phenomenon important for the direct interaction of NDUFC2 with the mtDNA-encoded complex I subunit ND4. This finding underlines the importance of mitonuclear coevolution to physical interactions between mtDNA and nuclear DNA-encoded factors. Nevertheless, it remains unclear whether this interaction is important for the stability and activity of complex I. Here, we show that siRNA silencing of NDUFC2 reduced growth of human D-407 retinal pigment epithelial cells, significantly diminished mitochondrial membrane potential, and interfered with complex I integrity. Moreover, site-directed mutagenesis of a positively selected amino acid in NDUFC2 significantly interfered with the interaction of NDUFC2 with its mtDNA-encoded partner ND4. Finally, we show that a genotype combination involving this amino acid (NDUFC2 residue 46) and the mtDNA haplogroup HV likely altered susceptibility to type 2 diabetes mellitus in Ashkenazi Jews. Therefore, mitonuclear coevolution is important for maintaining mitonuclear factor interactions, OXPHOS, and for human health.

Mito-nuclear co-evolution: the positive and negative sides of functional ancient mutations

Most cell functions are carried out by interacting factors, thus underlying the functional importance of genetic interactions between genes, termed epistasis. Epistasis could be under strong selective pressures especially in conditions where the mutation rate of one of the interacting partners notably differs from the other. Accordingly, the order of magnitude higher mitochondrial DNA (mtDNA) mutation rate as compared to the nuclear DNA (nDNA) of all tested animals, should influence systems involving mitochondrial-nuclear (mito-nuclear) interactions. Such is the case of the energy producing oxidative phosphorylation (OXPHOS) and mitochondrial translational machineries which are comprised of factors encoded by both the mtDNA and the nDNA. Additionally, the mitochondrial RNA transcription and mtDNA replication systems are operated by nDNA-encoded proteins that bind mtDNA regulatory elements. As these systems are central to cell life there is strong selection toward mito-nuclear co-evolution to maintain their function. However, it is unclear whether (A) mito-nuclear co-evolution befalls only to retain mitochondrial functions during evolution or, also, (B) serves as an adaptive tool to adjust for the evolving energetic demands as species’ complexity increases. As the first step to answer these questions we discuss evidence of both negative and adaptive (positive) selection acting on the mtDNA and nDNA-encoded genes and the effect of both types of selection on mito-nuclear interacting factors. Emphasis is given to the crucial role of recurrent ancient (nodal) mutations in such selective events. We apply this point-of-view to the three available types of mito-nuclear co-evolution: protein–protein (within the OXPHOS system), protein-RNA (mainly within the mitochondrial ribosome), and protein-DNA (at the mitochondrial replication and transcription machineries).

Functional Recurrent Mutations in the Human Mitochondrial Phylogeny - Dual Roles in Evolution and Disease

Mutations frequently reoccur in the human mitochondrial DNA (mtDNA). However, it is unclear whether recurrent mtDNA nodal mutations (RNMs), that is, recurrent mutations in stems of unrelated phylogenetic nodes, are functional and hence selectively constrained. To answer this question, we performed comprehensive parsimony and maximum likelihood analyses of 9,868 publicly available whole human mtDNAs revealing 1,606 single nodal mutations (SNMs) and 679 RNMs. We then evaluated the potential functionality of synonymous, nonsynonymous and RNA SNMs and RNMs. For synonymous mutations, we have implemented the Codon Adaptation Index. For nonsynonymous mutations, we assessed evolutionary conservation, and employed previously described pathogenicity score assessment tools. For RNA genes’ mutations, we designed a bioinformatic tool which compiled evolutionary conservation and potential effect on RNA structure. While comparing the functionality scores of nonsynonymous and RNA SNMs and RNMs with those of disease-causing mtDNA mutations, we found significant difference (P < 0.001). However, 24 RNMs and 67 SNMs had comparable values with disease-causing mutations reflecting their potential function thus being the best candidates to participate in adaptive events of unrelated lineages. Strikingly, some functional RNMs occurred in unrelated mtDNA lineages that independently altered susceptibility to the same diseases, thus suggesting common functionality. To our knowledge, this is the most comprehensive analysis of selective signatures in the mtDNA not only within proteins but also within RNA genes. For the first time, we discover virtually all positively selected RNMs in our phylogeny while emphasizing their dual role in past evolutionary events and in disease today.

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.

Mitochondrial Involvement in Vertebrate Speciation? The Case of Mito-nuclear Genetic Divergence in Chameleons.

Compatibility between the nuclear (nDNA) and mitochondrial (mtDNA) genomes is important for organismal health. However, its significance for major evolutionary processes such as speciation is unclear, especially in vertebrates. We previously identified a sharp mtDNA-specific sequence divergence between morphologically indistinguishable chameleon populations (Chamaeleo chamaeleon recticrista) across an ancient Israeli marine barrier (Jezreel Valley). Because mtDNA introgression and gender-based dispersal were ruled out, we hypothesized that mtDNA spatial division was maintained by mito-nuclear functional compensation. Here, we studied RNA-seq generated from each of ten chameleons representing the north and south populations and identified candidate nonsynonymous substitutions (NSSs) matching the mtDNA spatial distribution. The most prominent NSS occurred in 14 nDNA-encoded mitochondrial proteins. Increased chameleon sample size (N = 70) confirmed the geographic differentiation in POLRMT, NDUFA5, ACO1, LYRM4, MARS2, and ACAD9. Structural and functionality evaluation of these NSSs revealed high functionality. Mathematical modeling suggested that this mito-nuclear spatial divergence is consistent with hybrid breakdown. We conclude that our presented evidence and mathematical model underline mito-nuclear interactions as a likely role player in incipient speciation in vertebrates.

Improved Structures for Data Collection in Wireless Sensor Networks

In this paper we consider the problem of efficient data gathering in sensor networks for arbitrary sensor node deployments. The efficiency of the solution is measured by a number of criteria: total energy consumption, total transport capacity, latency and quality of the transmissions. We present a number of different constructions with various tradeoffs between aforementioned parameters. We provide theoretical performance analysis for our approaches, present their distributed implementation and discuss the different aspects of using each. We show that in many cases our output-sensitive approximation solution performs better than the currently known best results for sensor networks. Our simulation results validate the theoretical findings.

Transcription Factors Bind Negatively Selected Sites within Human mtDNA Genes

Transcription of mitochondrial DNA (mtDNA)-encoded genes is thought to be regulated by a handful of dedicated transcription factors (TFs), suggesting that mtDNA genes are separately regulated from the nucleus. However, several TFs, with known nuclear activities, were found to bind mtDNA and regulate mitochondrial transcription. Additionally, mtDNA transcriptional regulatory elements, which were proved important in vitro, were harbored by a deletion that normally segregated among healthy individuals. Hence, mtDNA transcriptional regulation is more complex than once thought. Here, by analyzing ENCODE chromatin immunoprecipitation sequencing (ChIP-seq) data, we identified strong binding sites of three bona fide nuclear TFs (c-Jun, Jun-D, and CEBPb) within human mtDNA protein-coding genes. We validated the binding of two TFs by ChIP-quantitative polymerase chain reaction (c-Jun and Jun-D) and showed their mitochondrial localization by electron microscopy and subcellular fractionation. As a step toward investigating the functionality of these TF-binding sites (TFBS), we assessed signatures of selection. By analyzing 9,868 human mtDNA sequences encompassing all major global populations, we recorded genetic variants in tips and nodes of mtDNA phylogeny within the TFBS. We next calculated the effects of variants on binding motif prediction scores. Finally, the mtDNA variation pattern in predicted TFBS, occurring within ChIP-seq negative-binding sites, was compared with ChIP-seq positive-TFBS (CPR). Motifs within CPRs of c-Jun, Jun-D, and CEBPb harbored either only tip variants or their nodal variants retained high motif prediction scores. This reflects negative selection within mtDNA CPRs, thus supporting their functionality. Hence, human mtDNA-coding sequences may have dual roles, namely coding for genes yet possibly also possessing regulatory potential.

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.

The genomic landscape of evolutionary convergence in mammals, birds and reptiles

Many lineage-defining (nodal) mutations possess high functionality. However, differentiating adaptive nodal mutations from those that are functionally compensated remains challenging. To address this challenge, we identified functional nodal mutations (fNMs) in ~3,400 nuclear DNA (nDNA) and 4 mitochondrial DNA (mtDNA) protein structures from 91 and 1,003 species, respectively, representing the entire mammalian, bird and reptile phylogeny. A screen for candidate compensatory mutations among co-occurring amino acid changes in close structural proximity revealed that such compensated fNMs encompass 37% and 27% of the mtDNA and nDNA datasets, respectively. Analysis of the remaining (non-compensated) mutations, which are enriched for adaptive mutations, showed that birds and mammals share most such recurrent fNMs (N = 51). Among the latter, we discovered mutations in thermoregulation-related genes. These represent the best candidates to explain the molecular basis of convergent body thermoregulation in birds and mammals. Our analysis reveals the landscape of possible mutational compensation and convergence in amniote phylogeny.

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.