Comparative analysis of mitochondrial genomes in different species reveals the evolution of G-quadruplexes

G-quadruplexes (G4s) are noncanonical structures that can form in the genomes of a range of organisms and are known to play various roles in cellular function. G4s can also form in mitochondrial DNA (mtDNA) because of their high guanine content, and these G4s may play roles in regulating gene expression, DNA replication, and genome stability. However, little is known regarding the evolution and dissemination of G4s in mitochondria. Here we analyzed the potential G4-forming sequences in mtDNA of 16 species from various families and demonstrated that the heavy strand of mtDNA of higher-order organisms contained higher levels of G4 regions than that of lower-order organisms. Analysis of the codons in the light strand revealed enrichment of guanine/cytosine-rich regions in higher eukaryotes and of adenine/thymidine-rich regions in lower-order organisms. Our study showed the diversity of G4s in species ranging from lower to higher orders. In particular, mammals such as humans, chimpanzees, and monkeys display a greater number of G4s than lower-order organisms. These potentially play a role in a range of cellular functions and assist in the evolution of higher organisms.

The mitochondrial single-stranded DNA binding protein is essential for initiation of mtDNA replication

We report a role for the mitochondrial single-stranded DNA binding protein (mtSSB) in regulating mitochondrial DNA (mtDNA) replication initiation in mammalian mitochondria. Transcription from the light-strand promoter (LSP) is required both for gene expression and for generating the RNA primers needed for initiation of mtDNA synthesis. In the absence of mtSSB, transcription from LSP is strongly up-regulated, but no replication primers are formed. Using deep sequencing in a mouse knockout model and biochemical reconstitution experiments with pure proteins, we find that mtSSB is necessary to restrict transcription initiation to optimize RNA primer formation at both origins of mtDNA replication. Last, we show that human pathological versions of mtSSB causing severe mitochondrial disease cannot efficiently support primer formation and initiation of mtDNA replication.

Mitochondrial genome of Chinese grass shrimp, Palaemonetes sinensis and comparison with other Palaemoninae species

The mitogenome of Chinese grass shrimp, Palaemonetes sinensis, was determined through Illumina sequencing, and the basic characteristics and gene arrangement were analyzed. The mitogenome of P. sinensis was 15955 bp in length, consisting of 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes and one control region, with tightly packed. 33 of these genes were encoded on the heavy strand, and the remainders encoded on the light strand. The composition of P. sinensis mitogenome presented a strong A + T bias, which account for 66.7%. All PCGs were initiated by a canonical ATN codon, except nad5, which was initiated by GTG. The termination codons of the PCGs were TAA, TAG and T–. The secondary structures of 22 tRNAs of P. sinensis had the typical clover structure, except of trnS1 owing to the lack of dihydroxyuridine (DHU) arm. Gene order comparison of P. sinensis and previously-sequenced Palaemoninae revealed a unique translocation between trnT and trnP in Macrobrachium. The phylogenetic analyses showed that three Exopalaemon species formed a monophyletic group and then clustered with two Palaemon species and P. sinensis successively whereas Macrobrachium clustered with Palaemon capensis in the other clade.

Hypertension-associated mitochondrial DNA 4401A>G mutation caused the aberrant processing of tRNAMet, all 8 tRNAs and ND6 mRNA in the light-strand transcript

Mitochondrial tRNA processing defects were associated with human diseases but their pathophysiology remains elusively. The hypertension-associated m.4401A>G mutation resided at a spacer between mitochondrial tRNAMet and tRNAGln genes. An in vitro processing experiment revealed that the m.4401A>G mutation caused 59% and 69% decreases in the 5′ end processing efficiency of tRNAGln and tRNAMet precursors, catalyzed by RNase P, respectively. Using human umbilical vein endothelial cells-derived cybrids, we demonstrated that the m.4401A>G mutation caused the decreases of all 8 tRNAs and ND6 and increases of longer and uncleaved precursors from the Light-strand transcript. Conversely, the m.4401A>G mutation yielded the reduced levels of tRNAMet level but did not change the levels of other 13 tRNAs, 12 mRNAs including ND1, 12S rRNA and 16S rRNA from the Heavy-strand transcript. These implicated the asymmetrical processing mechanisms of H-strand and L-strand polycistronic transcripts. The tRNA processing defects play the determined roles in the impairing mitochondrial translation, respiratory deficiency, diminishing membrane potential, increasing production of reactive oxygen species and altering autophagy. Furthermore, the m.4401A>G mutation altered the angiogenesis, evidenced by aberrant wound regeneration and weaken tube formation in mutant cybrids. Our findings provide new insights into the pathophysiology of hypertension arising from mitochondrial tRNA processing defects.

Low-level mitochondrial heteroplasmy modulates DNA replication, glucose metabolism and lifespan in mice

SummaryMutations in mitochondrial DNA (mtDNA) lead to heteroplasmy, i.e. the intracellular coexistence of wild-type and mutant mtDNA strands, which impact a wide spectrum of diseases but also physiological processes, including endurance exercise performance in athletes. However, the phenotypic consequences of limited levels of naturally-arising heteroplasmy have not been experimentally studied to date. We hence generated a conplastic mouse strain carrying the mitochondrial genome of a AKR/J mouse strain (B6-mtAKR) together with a C57BL/6J nuclear genomic background, leading to >20% heteroplasmy in the origin of light-strand DNA replication (OriL). These conplastic mice demonstrate a shorter lifespan as well as dysregulation of multiple metabolic pathways, culminating in impaired glucose metabolism, compared to wild-type C57BL/6J mice carrying lower levels of heteroplasmy. Our results indicate that physiologically relevant differences in mtDNA heteroplasmy levels at a single, functionally important site impair metabolic health and lifespan in mice.HighlightsWe identify heteroplasmy of the adenine-repeat variation (9 to 13A) in nt5172 in the origin of light-strand DNA replication (OriL) in inbred mice.B6-mtAKR mice carry >20% 12A heteroplasmy in the OriL, while B6 mice carry only ∼ 10% heteroplasmy.The level of 12A heteroplasmy correlates to mtDNA copy number, glucose metabolism, and lifespan in mice.Given the established role of mtDNA heteroplasmy in regards to endurance exercise performance in athletes, these findings may impact our understanding of metabolism and aging in humans.

Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter

Human mtDNA contains three promoters, suggesting a need for differential expression of the mitochondrial genome. Studies of mitochondrial transcription have used a reductionist approach, perhaps masking differential regulation. Here we evaluate transcription from light-strand (LSP) and heavy-strand (HSP1) promoters using templates that mimic their natural context. These studies reveal sequences upstream, hypervariable in the human population (HVR3), and downstream of the HSP1 transcription start site required for maximal yield. The carboxy-terminal tail of TFAM is essential for activation of HSP1 but not LSP. Images of the template obtained by atomic force microscopy show that TFAM creates loops in a discrete region, the formation of which correlates with activation of HSP1; looping is lost in tail-deleted TFAM. Identification of HVR3 as a transcriptional regulatory element may contribute to between-individual variability in mitochondrial gene expression. The unique requirement of HSP1 for the TFAM tail may enable its regulation by post-translational modifications.

Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter

Human mtDNA contains three promoters, suggesting a need for differential expression of the mitochondrial genome. Studies of mitochondrial transcription have used a reductionist approach, perhaps masking differential regulation. Here we evaluate transcription from light-strand (LSP) and heavy-strand (HSP1) promoters using templates that mimic their natural context. These studies reveal sequences upstream, hypervariable in the human population (HVR3), and downstream of the HSP1 transcription start site required for maximal yield. The carboxy-terminal tail of TFAM is essential for activation of HSP1 but not LSP. Images of the template obtained by atomic force microscopy show that TFAM creates loops in a discrete region, the formation of which correlates with activation of HSP1; looping is lost in tail-deleted TFAM. Identification of HVR3 as a transcriptional regulatory element may contribute to between-individual variability in mitochondrial gene expression. The unique requirement of HSP1 for the TFAM tail may enable its regulation by post-translational modifications.

Comparative mitogenomic analyses reveal cryptic diversity of the bryozoan Bugula neritina Linnaeus, 1758, in the Yellow Sea

The bryozoan Bugula neritina Linnaeus, 1758, is known to be a complex of three cryptic species, namely Types S, D and N. In the present study, we determined the mitochondrial genomic features of B. neritina sampled from Qingdao (QD), China, and compared them with those of the genome reported for a specimen sampled from Taean Gun (TG), South Korea. The B. neritina QD mitochondrial genome has a duplication of trnL2 and lacks trnV compared with B. neritina TG. Five tRNAs (trnL1, trnA, trnE, trnY and trnV) are encoded on the light-strand of B. neritina TG mitochondrial genome, but only one tRNA (trnA) is identified on the B. neritina QD mitochondrial light strand. In contrast to the B. neritina TG mitochondrial genome, deletion of trnV and duplication of trnL2 are identified in the B. neritina QD mtDNA, and three tRNAs (trnE, trnL1 and trnY) exhibit translocation and inversion. The genetic distance in 12 protein-coding genes (PCGs) (amino acids) between the two B. neritina was 0.079, which is higher than interspecific values of 10 lophotrochozoan genera selected for comparison. All these results from comparison between the two B. neritina clearly indicate that they are genetically distinct species. Phylogenetic analysis based on cox1 and lrRNA sequences suggested that B. neritina TG belongs to the widely distributed Type S and B. neritina QD represents a new cryptic type closely related to Type N. This new type is designated as Type Y, for its occurrence in the Yellow Sea. The geographical range of the different types of B. neritina awaits further studies.