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1.  Transposable elements and circular DNAs 
Mobile Genetic Elements  2016;6(6):453-62.
Circular DNAs are extra-chromosomal fragments that become circularized by genomic recombination events. We have recently shown that yeast LTR elements generate circular DNAs through recombination events between their flanking long terminal repeats (LTRs). Similarly, circular DNAs can be generated by recombination between LTRs residing at different genomic loci, in which case the circular DNA will contain the intervening sequence. In yeast, this can result in gene copy number variations when circles contain genes and origins of replication. Here, I speculate on the potential and implications of circular DNAs generated through recombination between human transposable elements.
PMCID: PMC5173269  PMID: 28090380
Alu elements; circular DNAs; eccDNAs; evolution; genome; human; yeast
2.  A Phenotype-Driven Approach to Generate Mouse Models with Pathogenic mtDNA Mutations Causing Mitochondrial Disease 
Cell Reports  2016;16(11):2980-2990.
Mutations of mtDNA are an important cause of human disease, but few animal models exist. Because mammalian mitochondria cannot be transfected, the development of mice with pathogenic mtDNA mutations has been challenging, and the main strategy has therefore been to introduce mutations found in cell lines into mouse embryos. Here, we describe a phenotype-driven strategy that is based on detecting clonal expansion of pathogenic mtDNA mutations in colonic crypts of founder mice derived from heterozygous mtDNA mutator mice. As proof of concept, we report the generation of a mouse line transmitting a heteroplasmic pathogenic mutation in the alanine tRNA gene of mtDNA displaying typical characteristics of classic mitochondrial disease. In summary, we describe a straightforward and technically simple strategy based on mouse breeding and histology to generate animal models of mtDNA-mutation disease, which will be of great importance for studies of disease pathophysiology and preclinical treatment trials.
Graphical Abstract
Image 1
•We present a method to isolate and identify pathogenic mtDNA mutations in mice•We describe a mouse with a pathogenic mutation in the mitochondrial tRNAALA gene•The mice display disrupted mitochondrial translation as a result of the mutation•The mice display molecular and histochemical symptoms of human mitochondrial disease
Kauppila et al. describe a phenotype-based screen in live mice to generate mouse models with pathogenic mtDNA mutations. As proof of concept, they present a mouse with a mutation in the mitochondrial tRNAALA gene that displays molecular and histochemical symptoms of human mitochondrial disease.
PMCID: PMC5039181  PMID: 27626666
3.  COX7A2L is a mitochondrial complex III-binding protein that stabilizes the III2+IV supercomplex without affecting respirasome formation 
Cell reports  2016;16(9):2387-2398.
Mitochondrial respiratory chain (MRC) complexes I, III and IV associate into a variety of supramolecular structures known as supercomplexes and respirasomes. While COX7A2L was originally described as a supercomplex-specific factor responsible for the dynamic association of complex IV into these structures to adapt MRC function to metabolic variations, this role has been disputed. Here we further examine the functional significance of COX7A2L in the structural organization of the mammalian respiratory chain. As in the mouse, human COX7A2L binds primarily to free mitochondrial complex III and to a minor extent to complex IV to specifically promote the stabilization of the III2+IV supercomplex without affecting respirasome formation. Furthermore, COX7A2L does not affect the biogenesis, stabilization and function of the individual OXPHOS complexes. These data show that independent regulatory mechanisms for the biogenesis and turnover of different MRC supercomplex structures co-exist.
Graphical abstract
PMCID: PMC5007171  PMID: 27545886
4.  POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA 
Science Advances  2016;2(8):e1600963.
Mitochondrial transcription for replication primer formation has priority over gene expression at low POLRMT levels.
Mitochondria are vital in providing cellular energy via their oxidative phosphorylation system, which requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes (mtDNA). Transcription of the circular mammalian mtDNA depends on a single mitochondrial RNA polymerase (POLRMT). Although the transcription initiation process is well understood, it is debated whether POLRMT also serves as the primase for the initiation of mtDNA replication. In the nucleus, the RNA polymerases needed for gene expression have no such role. Conditional knockout of Polrmt in the heart results in severe mitochondrial dysfunction causing dilated cardiomyopathy in young mice. We further studied the molecular consequences of different expression levels of POLRMT and found that POLRMT is essential for primer synthesis to initiate mtDNA replication in vivo. Furthermore, transcription initiation for primer formation has priority over gene expression. Surprisingly, mitochondrial transcription factor A (TFAM) exists in an mtDNA-free pool in the Polrmt knockout mice. TFAM levels remain unchanged despite strong mtDNA depletion, and TFAM is thus protected from degradation of the AAA+ Lon protease in the absence of POLRMT. Last, we report that mitochondrial transcription elongation factor may compensate for a partial depletion of POLRMT in heterozygous Polrmt knockout mice, indicating a direct regulatory role of this factor in transcription. In conclusion, we present in vivo evidence that POLRMT has a key regulatory role in the replication of mammalian mtDNA and is part of a transcriptional mechanism that provides a switch between primer formation for mtDNA replication and mitochondrial gene expression.
PMCID: PMC4975551  PMID: 27532055
Mitochondria; mitochondrial RNA polymerase; mitochondrial gene expression; mtDNA; mtDNA replication; 7S RNA; light strand promoter; mtDNA-free TFAM pool; twinkle; POLRMT knockout mouse
5.  High diversity of picornaviruses in rats from different continents revealed by deep sequencing 
Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus norvegicus (R. norvegicus) is a known reservoir for important zoonotic pathogens. Transmission may be direct via contact with the animal, for example, through exposure to its faecal matter, or indirectly mediated by arthropod vectors. Here we investigated the viral content in rat faecal matter (n=29) collected from two continents by analyzing 2.2 billion next-generation sequencing reads derived from both DNA and RNA. Among other virus families, we found sequences from members of the Picornaviridae to be abundant in the microbiome of all the samples. Here we describe the diversity of the picornavirus-like contigs including near-full-length genomes closely related to the Boone cardiovirus and Theiler's encephalomyelitis virus. From this study, we conclude that picornaviruses within R. norvegicus are more diverse than previously recognized. The virome of R. norvegicus should be investigated further to assess the full potential for zoonotic virus transmission.
PMCID: PMC5034103  PMID: 27530749
cardiovirus; metagenomics; picornavirus; Rattus norvegicus; sequencing; viral discovery
6.  Maternal exposure to diluted diesel engine exhaust alters placental function and induces intergenerational effects in rabbits 
Airborne pollution is a rising concern in urban areas. Epidemiological studies in humans and animal experiments using rodent models indicate that gestational exposure to airborne pollution, in particular diesel engine exhaust (DE), reduces birth weight, but effects depend on exposure duration, gestational window and nanoparticle (NP) concentration. Our aim was to evaluate the effects of gestational exposure to diluted DE on feto-placental development in a rabbit model.
Pregnant females were exposed to diluted (1 mg/m3), filtered DE (NP diameter ≈ 69 nm) or clean air (controls) for 2 h/day, 5 days/week by nose-only exposure (total exposure: 20 days in a 31-day gestation).
DE exposure induced early signs of growth retardation at mid gestation with decreased head length (p = 0.04) and umbilical pulse (p = 0.018). Near term, fetal head length (p = 0.029) and plasma insulin and IGF1 concentrations (p = 0.05 and p = 0.019) were reduced. Placental function was also affected, with reduced placental efficiency (fetal/placental weight) (p = 0.049), decreased placental blood flow (p = 0.009) and fetal vessel volume (p = 0.002). Non-aggregated and “fingerprint” NP were observed at various locations, in maternal blood space, in trophoblastic cells and in the fetal blood, demonstrating transplacental transfer. Adult female offspring were bred with control males. Although fetoplacental biometry was not affected near term, second generation fetal metabolism was modified by grand-dam exposure with decreased plasma cholesterol (p = 0.008) and increased triglyceride concentrations (p = 0.015).
Repeated daily gestational exposure to DE at levels close to urban pollution can affect feto-placental development in the first and second generation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12989-016-0151-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4962477  PMID: 27460165
7.  Respiratory Effects of Sarafotoxins from the Venom of Different Atractaspis Genus Snake Species 
Toxins  2016;8(7):215.
Sarafotoxins (SRTX) are endothelin-like peptides extracted from the venom of snakes belonging to the Atractaspididae family. A recent in vivo study on anesthetized and ventilated animals showed that sarafotoxin-b (SRTX-b), extracted from the venom of Atractaspis engaddensis, decreases cardiac output by inducing left ventricular dysfunction while sarafotoxin-m (SRTX-m), extracted from the venom of Atractaspis microlepidota microlepidota, induces right ventricular dysfunction with increased airway pressure. The aim of the present experimental study was to compare the respiratory effects of SRTX-m and SRTX-b. Male Wistar rats were anesthetized, tracheotomized and mechanically ventilated. They received either a 1 LD50 IV bolus of SRTX-b (n = 5) or 1 LD50 of SRTX-m (n = 5). The low-frequency forced oscillation technique was used to measure respiratory impedance. Airway resistance (Raw), parenchymal damping (G) and elastance (H) were determined from impedance data, before and 5 min after SRTX injection. SRTX-m and SRTX-b injections induced acute hypoxia and metabolic acidosis with an increased anion gap. Both toxins markedly increased Raw, G and H, but with a much greater effect of SRTX-b on H, which may have been due to pulmonary edema in addition to bronchoconstriction. Therefore, despite their structural analogy, these two toxins exert different effects on respiratory function. These results emphasize the role of the C-terminal extension in the in vivo effect of these toxins.
PMCID: PMC4963848  PMID: 27409637
Atractaspis; long sarafotoxins; respiratory mechanics; forced oscillation technique; hysterisivity
8.  Propionibacterium acnes: Disease-Causing Agent or Common Contaminant? Detection in Diverse Patient Samples by Next-Generation Sequencing 
Journal of Clinical Microbiology  2016;54(4):980-987.
Propionibacterium acnes is the most abundant bacterium on human skin, particularly in sebaceous areas. P. acnes is suggested to be an opportunistic pathogen involved in the development of diverse medical conditions but is also a proven contaminant of human clinical samples and surgical wounds. Its significance as a pathogen is consequently a matter of debate. In the present study, we investigated the presence of P. acnes DNA in 250 next-generation sequencing data sets generated from 180 samples of 20 different sample types, mostly of cancerous origin. The samples were subjected to either microbial enrichment, involving nuclease treatment to reduce the amount of host nucleic acids, or shotgun sequencing. We detected high proportions of P. acnes DNA in enriched samples, particularly skin tissue-derived and other tissue samples, with the levels being higher in enriched samples than in shotgun-sequenced samples. P. acnes reads were detected in most samples analyzed, though the proportions in most shotgun-sequenced samples were low. Our results show that P. acnes can be detected in practically all sample types when molecular methods, such as next-generation sequencing, are employed. The possibility of contamination from the patient or other sources, including laboratory reagents or environment, should therefore always be considered carefully when P. acnes is detected in clinical samples. We advocate that detection of P. acnes always be accompanied by experiments validating the association between this bacterium and any clinical condition.
PMCID: PMC4809928  PMID: 26818667
9.  Identification of Known and Novel Recurrent Viral Sequences in Data from Multiple Patients and Multiple Cancers 
Viruses  2016;8(2):53.
Virus discovery from high throughput sequencing data often follows a bottom-up approach where taxonomic annotation takes place prior to association to disease. Albeit effective in some cases, the approach fails to detect novel pathogens and remote variants not present in reference databases. We have developed a species independent pipeline that utilises sequence clustering for the identification of nucleotide sequences that co-occur across multiple sequencing data instances. We applied the workflow to 686 sequencing libraries from 252 cancer samples of different cancer and tissue types, 32 non-template controls, and 24 test samples. Recurrent sequences were statistically associated to biological, methodological or technical features with the aim to identify novel pathogens or plausible contaminants that may associate to a particular kit or method. We provide examples of identified inhabitants of the healthy tissue flora as well as experimental contaminants. Unmapped sequences that co-occur with high statistical significance potentially represent the unknown sequence space where novel pathogens can be identified.
PMCID: PMC4776208  PMID: 26907326
sequence clustering; taxonomic characterisation; novel sequence identification; next generation sequencing; cancer causing viruses; oncoviruses; assay contamination
10.  Blacktip reef sharks (Carcharhinus melanopterus) show high capacity for wound healing and recovery following injury 
Conservation Physiology  2015;3(1):cov062.
Wounds heal quickly in blacktip reef sharks. In captive neonates, umbilical scars heal within weeks. In mature individuals, predator wounds and/or telemetry tag surgical procedure wounds heal in weeks to months. This information is important for monitoring populations and assessing habitat degradation impacts and interactions with fishing gear on sharks.
Wound healing is important for sharks from the earliest life stages, for example, as the ‘umbilical scar’ in viviparous species heals, and throughout adulthood, when sharks can incur a range of external injuries from natural and anthropogenic sources. Despite anecdotal accounts of rapid healing in elasmobranchs, data regarding recovery and survival of individuals from different wound or injury types has not been systematically collected. The present study documented: (i) ‘umbilical scar’ healing in wild-caught, neonatal blacktip reef sharks while being reared for 30 days in flow-through laboratory aquaria in French Polynesia; (ii) survival and recovery of free-swimming blacktip reef sharks in Australia and French Polynesia following a range of injuries; and (iii) long-term survival following suspected shark-finning activities. Laboratory monitoring, tag-recapture records, telemetry data and photo-identification records suggest that blacktip reef sharks have a high capacity to survive and recover from small or even large and severe wounds. Healing rates, recovery and survival are important factors to consider when assessing impacts of habitat degradation and fishing stress on shark populations. The present study suggests that individual survival may depend more on handling practices and physiological stress rather than the extent of physical injury. These observations also contribute to discussions regarding the ethics of tagging practices used in elasmobranch research and provide baseline healing rates that may increase the accuracy in estimating reproductive timing inferred from mating scars and birth dates for neonatal sharks based on umbilical scar healing status.
PMCID: PMC4778477  PMID: 27293741
Coral reefs; elasmobranchs; fish; fisheries; French Polynesia; Great Barrier Reef
11.  Formation of Extrachromosomal Circular DNA from Long Terminal Repeats of Retrotransposons in Saccharomyces cerevisiae 
G3: Genes|Genomes|Genetics  2015;6(2):453-462.
Extrachromosomal circular DNA (eccDNA) derived from chromosomal Ty retrotransposons in yeast can be generated in multiple ways. Ty eccDNA can arise from the circularization of extrachromosomal linear DNA during the transpositional life cycle of retrotransposons, or from circularization of genomic Ty DNA. Circularization may happen through nonhomologous end-joining (NHEJ) of long terminal repeats (LTRs) flanking Ty elements, by Ty autointegration, or by LTR–LTR recombination. By performing an in-depth investigation of sequence reads stemming from Ty eccDNAs obtained from populations of Saccharomyces cerevisiae S288c, we find that eccDNAs predominantly correspond to full-length Ty1 elements. Analyses of sequence junctions reveal no signs of NHEJ or autointegration events. We detect recombination junctions that are consistent with yeast Ty eccDNAs being generated through recombination events within the genome. This opens the possibility that retrotransposable elements could move around in the genome without an RNA intermediate directly through DNA circularization.
PMCID: PMC4751563  PMID: 26681518
transposable elements; long terminal repeats; yeast; recombination; circular DNA
12.  Rescue of primary ubiquinone deficiency due to a novel COQ7 defect using 2,4–dihydroxybensoic acid 
Journal of Medical Genetics  2015;52(11):779-783.
Coenzyme Q is an essential mitochondrial electron carrier, redox cofactor and a potent antioxidant in the majority of cellular membranes. Coenzyme Q deficiency has been associated with a range of metabolic diseases, as well as with some drug treatments and ageing.
We used whole exome sequencing (WES) to investigate patients with inherited metabolic diseases and applied a novel ultra-pressure liquid chromatography—mass spectrometry approach to measure coenzyme Q in patient samples.
We identified a homozygous missense mutation in the COQ7 gene in a patient with complex mitochondrial deficiency, resulting in severely reduced coenzyme Q levels We demonstrate that the coenzyme Q analogue 2,4-dihydroxybensoic acid (2,4DHB) was able to specifically bypass the COQ7 deficiency, increase cellular coenzyme Q levels and rescue the biochemical defect in patient fibroblasts.
We report the first patient with primary coenzyme Q deficiency due to a homozygous COQ7 mutation and a potentially beneficial treatment using 2,4DHB.
PMCID: PMC4680133  PMID: 26084283
Metabolic disorders; Molecular genetics
13.  The Genomic and Phenotypic Diversity of Schizosaccharomyces pombe 
Nature genetics  2015;47(3):235-241.
Natural variation within species reveals aspects of genome evolution and function. The fission yeast Schizosaccharomyces pombe is an important model for eukaryotic biology, but researchers typically use one standard laboratory strain. To extend the utility of this model, we surveyed the genomic and phenotypic variation in 161 natural isolates. We sequenced the genomes of all strains, revealing moderate genetic diversity (π = 3 ×10−3) and weak global population structure. We estimate that dispersal of S. pombe began within human antiquity (~340 BCE), and ancestors of these strains reached the Americas at ~1623 CE. We quantified 74 traits, revealing substantial heritable phenotypic diversity. We conducted 223 genome-wide association studies, with 89 traits showing at least one association. The most significant variant for each trait explained 22% of variance on average, with indels having higher effects than SNPs. This analysis presents a rich resource to examine genotype-phenotype relationships in a tractable model.
PMCID: PMC4645456  PMID: 25665008
14.  Characterizing novel endogenous retroviruses from genetic variation inferred from short sequence reads 
Scientific Reports  2015;5:15644.
From Illumina sequencing of DNA from brain and liver tissue from the lion, Panthera leo, and tumor samples from the pike-perch, Sander lucioperca, we obtained two assembled sequence contigs with similarity to known retroviruses. Phylogenetic analyses suggest that the pike-perch retrovirus belongs to the epsilonretroviruses, and the lion retrovirus to the gammaretroviruses. To determine if these novel retroviral sequences originate from an endogenous retrovirus or from a recently integrated exogenous retrovirus, we assessed the genetic diversity of the parental sequences from which the short Illumina reads are derived. First, we showed by simulations that we can robustly infer the level of genetic diversity from short sequence reads. Second, we find that the measures of nucleotide diversity inferred from our retroviral sequences significantly exceed the level observed from Human Immunodeficiency Virus infections, prompting us to conclude that the novel retroviruses are both of endogenous origin. Through further simulations, we rule out the possibility that the observed elevated levels of nucleotide diversity are the result of co-infection with two closely related exogenous retroviruses.
PMCID: PMC4616055  PMID: 26493184
15.  In Vivo Evaluation of Cervical Stiffness Evolution during Induced Ripening Using Shear Wave Elastography, Histology and 2 Photon Excitation Microscopy: Insight from an Animal Model 
PLoS ONE  2015;10(8):e0133377.
Prematurity affects 11% of the births and is the main cause of infant mortality. On the opposite case, the failure of induction of parturition in the case of delayed spontaneous birth is associated with fetal suffering. Both conditions are associated with precocious and/or delayed cervical ripening. Quantitative and objective information about the temporal evolution of the cervical ripening may provide a complementary method to identify cases at risk of preterm delivery and to assess the likelihood of successful induction of labour. In this study, the cervical stiffness was measured in vivo in pregnant sheep by using Shear Wave Elastography (SWE). This technique assesses the stiffness of tissue through the measurement of shear waves speed (SWS). In the present study, 9 pregnant ewes were used. Cervical ripening was induced at 127 days of pregnancy (term: 145 days) by dexamethasone injection in 5 animals, while 4 animals were used as control. Elastographic images of the cervix were obtained by two independent operators every 4 hours during 24 hours after injection to monitor the cervical maturation induced by the dexamethasone. Based on the measurements of SWS during vaginal ultrasound examination, the stiffness in the second ring of the cervix was quantified over a circular region of interest of 5 mm diameter. SWS was found to decrease significantly in the first 4–8 hours after dexamethasone compared to controls, which was associated with cervical ripening induced by dexamethasone (from 1.779 m/s ± 0.548 m/s, p < 0.0005, to 1.291 m/s ± 0.516 m/s, p < 0.000). Consequently a drop in the cervical elasticity was quantified too (from 9.5 kPa ± 0.9 kPa, p < 0.0005, to 5.0 kPa ± 0.8 kPa, p < 0.000). Moreover, SWE measurements were highly reproducible between both operators at all times. Cervical ripening induced by dexamethasone was confirmed by the significant increase in maternal plasma Prostaglandin E2 (PGE2), as evidenced by the assay of its metabolite PGEM. Histological analyses and two-photon excitation microscopy, combining both Second Harmonic Generation (SHG) and Two-photon Fluorescence microscopy (2PF) contrasts, were used to investigate, at the microscopic scale, the structure of cervical tissue. Results show that both collagen and 2PF-active fibrillar structures could be closely related to the mechanical properties of cervical tissue that are perceptible in elastography. In conclusion, SWE may be a valuable method to objectively quantify the cervical stiffness and as a complementary diagnostic tool for preterm birth and for labour induction success.
PMCID: PMC4552804  PMID: 26317774
16.  Investigation of Human Cancers for Retrovirus by Low-Stringency Target Enrichment and High-Throughput Sequencing 
Scientific Reports  2015;5:13201.
Although nearly one fifth of all human cancers have an infectious aetiology, the causes for the majority of cancers remain unexplained. Despite the enormous data output from high-throughput shotgun sequencing, viral DNA in a clinical sample typically constitutes a proportion of host DNA that is too small to be detected. Sequence variation among virus genomes complicates application of sequence-specific, and highly sensitive, PCR methods. Therefore, we aimed to develop and characterize a method that permits sensitive detection of sequences despite considerable variation. We demonstrate that our low-stringency in-solution hybridization method enables detection of <100 viral copies. Furthermore, distantly related proviral sequences may be enriched by orders of magnitude, enabling discovery of hitherto unknown viral sequences by high-throughput sequencing. The sensitivity was sufficient to detect retroviral sequences in clinical samples. We used this method to conduct an investigation for novel retrovirus in samples from three cancer types. In accordance with recent studies our investigation revealed no retroviral infections in human B-cell lymphoma cells, cutaneous T-cell lymphoma or colorectal cancer biopsies. Nonetheless, our generally applicable method makes sensitive detection possible and permits sequencing of distantly related sequences from complex material.
PMCID: PMC4541070  PMID: 26285800
17.  Mitofusin 2 is required to maintain mitochondrial coenzyme Q levels 
The Journal of Cell Biology  2015;208(4):429-442.
Mitofusin 2 plays an unexpected role in maintaining the terpenoid biosynthesis pathway and is necessary for mitochondrial coenzyme Q biosynthesis.
Mitochondria form a dynamic network within the cell as a result of balanced fusion and fission. Despite the established role of mitofusins (MFN1 and MFN2) in mitochondrial fusion, only MFN2 has been associated with metabolic and neurodegenerative diseases, which suggests that MFN2 is needed to maintain mitochondrial energy metabolism. The molecular basis for the mitochondrial dysfunction encountered in the absence of MFN2 is not understood. Here we show that loss of MFN2 leads to impaired mitochondrial respiration and reduced ATP production, and that this defective oxidative phosphorylation process unexpectedly originates from a depletion of the mitochondrial coenzyme Q pool. Our study unravels an unexpected and novel role for MFN2 in maintenance of the terpenoid biosynthesis pathway, which is necessary for mitochondrial coenzyme Q biosynthesis. The reduced respiratory chain function in cells lacking MFN2 can be partially rescued by coenzyme Q10 supplementation, which suggests a possible therapeutic strategy for patients with diseases caused by mutations in the Mfn2 gene.
PMCID: PMC4332246  PMID: 25688136
18.  SLIRP Regulates the Rate of Mitochondrial Protein Synthesis and Protects LRPPRC from Degradation 
PLoS Genetics  2015;11(8):e1005423.
We have studied the in vivo role of SLIRP in regulation of mitochondrial DNA (mtDNA) gene expression and show here that it stabilizes its interacting partner protein LRPPRC by protecting it from degradation. Although SLIRP is completely dependent on LRPPRC for its stability, reduced levels of LRPPRC persist in the absence of SLIRP in vivo. Surprisingly, Slirp knockout mice are apparently healthy and only display a minor weight loss, despite a 50–70% reduction in the steady-state levels of mtDNA-encoded mRNAs. In contrast to LRPPRC, SLIRP is dispensable for polyadenylation of mtDNA-encoded mRNAs. Instead, deep RNA sequencing (RNAseq) of mitochondrial ribosomal fractions and additional molecular analyses show that SLIRP is required for proper association of mRNAs to the mitochondrial ribosome and efficient translation. Our findings thus establish distinct functions for SLIRP and LRPPRC within the LRPPRC-SLIRP complex, with a novel role for SLIRP in mitochondrial translation. Very surprisingly, our results also demonstrate that mammalian mitochondria have a great excess of transcripts under basal physiological conditions in vivo.
Author Summary
Mitochondria provide most of the energy required for key metabolic and cellular processes that are essential for life. The biogenesis of the mitochondrial oxidative phosphorylation system, the site of energy conversion, is dependent on the coordinated expression of the mitochondrial and nuclear genomes. Mitochondrial gene expression is largely regulated at the post-transcriptional level by RNA-binding proteins, including the LRPPRC-SLIRP complex. It is still unclear how the proteins within this complex regulate mitochondrial RNA metabolism. Here, we have knocked out the Slirp gene in mice to dissect the individual roles of LRPPRC and SLIRP and provide further insights into the mechanisms governing post-transcriptional regulation of mitochondrial gene expression. LRPPRC is required for the maintenance of mitochondrial mRNA polyadenylation whereas SLIRP, by facilitating the presentation (or association) of mRNAs to the mitochondrial ribosome, regulates the rate of translation. In addition, we demonstrate that mitochondrial mRNAs in mammals are present in quantities that far exceed those needed to maintain normal physiology under basal conditions.
PMCID: PMC4527767  PMID: 26247782
19.  Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites 
eLife  null;4:e06974.
The eukaryotic phylum Apicomplexa encompasses thousands of obligate intracellular parasites of humans and animals with immense socio-economic and health impacts. We sequenced nuclear genomes of Chromera velia and Vitrella brassicaformis, free-living non-parasitic photosynthetic algae closely related to apicomplexans. Proteins from key metabolic pathways and from the endomembrane trafficking systems associated with a free-living lifestyle have been progressively and non-randomly lost during adaptation to parasitism. The free-living ancestor contained a broad repertoire of genes many of which were repurposed for parasitic processes, such as extracellular proteins, components of a motility apparatus, and DNA- and RNA-binding protein families. Based on transcriptome analyses across 36 environmental conditions, Chromera orthologs of apicomplexan invasion-related motility genes were co-regulated with genes encoding the flagellar apparatus, supporting the functional contribution of flagella to the evolution of invasion machinery. This study provides insights into how obligate parasites with diverse life strategies arose from a once free-living phototrophic marine alga.
eLife digest
Single-celled parasites cause many severe diseases in humans and animals. The apicomplexans form probably the most successful group of these parasites and include the parasites that cause malaria. Apicomplexans infect a broad range of hosts, including humans, reptiles, birds, and insects, and often have complicated life cycles. For example, the malaria-causing parasites spread by moving from humans to female mosquitoes and then back to humans.
Despite significant differences amongst apicomplexans, these single-celled parasites also share a number of features that are not seen in other living species. How and when these features arose remains unclear. It is known from previous work that apicomplexans are closely related to single-celled algae. But unlike apicomplexans, which depend on a host animal to survive, these algae live freely in their environment, often in close association with corals.
Woo et al. have now sequenced the genomes of two photosynthetic algae that are thought to be close living relatives of the apicomplexans. These genomes were then compared to each other and to the genomes of other algae and apicomplexans. These comparisons reconfirmed that the two algae that were studied were close relatives of the apicomplexans.
Further analyses suggested that thousands of genes were lost as an ancient free-living algae evolved into the apicomplexan ancestor, and further losses occurred as these early parasites evolved into modern species. The lost genes were typically those that are important for free-living organisms, but are either a hindrance to, or not needed in, a parasitic lifestyle. Some of the ancestor's genes, especially those that coded for the building blocks of flagella (structures which free-living algae use to move around), were repurposed in ways that helped the apicomplexans to invade their hosts. Understanding this repurposing process in greater detail will help to identify key molecules in these deadly parasites that could be targeted by drug treatments. It will also offer answers to one of the most fascinating questions in evolutionary biology: how parasites have evolved from free-living organisms.
PMCID: PMC4501334  PMID: 26175406
Chromera velia; Vitrella brassicaformis; evolution of parasitism; malaria; toxoplasmosis; other
20.  Short- versus Long-Sarafotoxins: Two Structurally Related Snake Toxins with Very Different in vivo Haemodynamic Effects 
PLoS ONE  2015;10(7):e0132864.
Sarafotoxin-m (24 amino acids) from the venom of Atractaspis microlepidota microlepidota was the first long-sarafotoxin to be identified, while sarafotoxin-b (21 aa) is a short-sarafotoxin from Atractaspis engaddensis. Despite the presence of three additional C-terminus residues in sarafotoxin-m, these two peptides display a high sequence homology and share similar three-dimensional structures. However, unlike sarafotoxin-b, sarafotoxin-m shows a very low in vitro affinity for endothelin receptors, but still has a very high in vivo toxicity in mammals, similar to that of sarafotoxin-b. We have previously demonstrated, in vitro, the crucial role of the C-terminus extension in terms of pharmacological profiles and receptor affinities of long- versus short-sarafotoxins. One possible hypothesis to explain the high in vivo toxicity of sarafotoxin-m could be that its C-terminus extension is processed in vivo, resulting in short-like sarafotoxin. To address this possibility, we investigated, in the present study, the in vivo cardiovascular effects of sarafotoxin-b, sarafotoxin-m and sarafotoxin-m−Cter (sarafotoxin-m without the C -terminus extension). Male Wistar rats were anaesthetised and mechanically ventilated. Invasive haemodynamic measurements and echocardiographic measurements of left and right ventricular function were performed. The rats were divided into four groups that respectively received intravenous injections of: saline, sarafotoxin-b (one LD50), sarafotoxin-m (one LD50) or sarafotoxin-m−Cter (one LD50). All measurements were performed at baseline, at 1 minute (+1) and at 6 minutes (+6) after injection. Results: Sarafotoxin-b and sarafotoxin-m-Cter decreased cardiac output and impaired left ventricle systolic and diastolic function, whilst sarafotoxin-m decreased cardiac output, increased airway pressures and led to acute right ventricular dilatation associated with a decreased tricuspid annulus peak systolic velocity. Sarafotoxin-b and sarafotoxin-m−Cter appear to exert toxic effects via impairment of left ventricular function, whilst sarafotoxin-m increases airway pressures and impairs right ventricular function. These results do not support the hypothesis of an in vivo processing of long sarafotoxins.
PMCID: PMC4503772  PMID: 26176218
21.  Draft Genome Sequence of the First Human Isolate of the Ruminant Pathogen Mycoplasma capricolum subsp. capricolum 
Genome Announcements  2015;3(3):e00583-15.
Mycoplasma capricolum subsp. capricolum is a well-known pathogen of small ruminants. A recent human case of septicemia involving this agent raised the question of its potential pathogenicity to humans. We present the first draft genome sequence of a human Mycoplasma capricolum subsp. capricolum isolate.
PMCID: PMC4472885  PMID: 26089408
22.  Crystallization of recombinant green mamba ρ-­Da1a toxin during a lyophilization procedure and its structure determination 
Acta Crystallographica Section F  2013;69(Pt 6):704-709.
Crystals of recombinantly expressed ρ-Da1a toxin from eastern green mamba that were suitable for structure determination were discovered on defrosting a partially lyophilized sample.
ρ-Da1a toxin from eastern green mamba (Dendroaspis angusticeps) venom is a polypeptide of 65 amino acids with a strong affinity for the G-protein-coupled α1A-adrenoceptor. This neurotoxin has been crystallized from resolubilized lyophilized powder, but the best crystals grew spontaneously during lyophilization. The crystals belonged to the trigonal space group P3121, with unit-cell parameters a = b = 37.37, c = 66.05 Å, and diffracted to 1.95 Å resolution. The structure solved by molecular replacement showed strong similarities to green mamba muscarinic toxins.
PMCID: PMC3668600  PMID: 23722859
lyophilization; three-finger fold toxin; weak data
23.  MICOS coordinates with respiratory complexes and lipids to establish mitochondrial inner membrane architecture 
eLife  null;4:e07739.
The conserved MICOS complex functions as a primary determinant of mitochondrial inner membrane structure. We address the organization and functional roles of MICOS and identify two independent MICOS subcomplexes: Mic27/Mic10/Mic12, whose assembly is dependent on respiratory complexes and the mitochondrial lipid cardiolipin, and Mic60/Mic19, which assembles independent of these factors. Our data suggest that MICOS subcomplexes independently localize to cristae junctions and are connected via Mic19, which functions to regulate subcomplex distribution, and thus, potentially also cristae junction copy number. MICOS subunits have non-redundant functions as the absence of both MICOS subcomplexes results in more severe morphological and respiratory growth defects than deletion of single MICOS subunits or subcomplexes. Mitochondrial defects resulting from MICOS loss are caused by misdistribution of respiratory complexes in the inner membrane. Together, our data are consistent with a model where MICOS, mitochondrial lipids and respiratory complexes coordinately build a functional and correctly shaped mitochondrial inner membrane.
eLife digest
Structures called mitochondria provide energy that cells need to live and grow. To do this, mitochondria convert energy stored within sugars and other carbon-rich compounds into the energy currency of cells, a molecule called adenosine triphosphate (called ATP for short). Defective mitochondria can cause cells to starve and also cause severe human diseases.
A double membrane surrounds each mitochondrion. The outer membrane allows proteins and other substances to enter, while the inner membrane is elaborately folded and contains several groups of proteins—or complexes—including the respiratory complexes that generate ATP. Proper inner membrane folding is critically important. The membrane folds are held in place by structures called cristae junctions, which may also help to restrict proteins to particular areas of the inner membrane.
A large inner membrane complex of proteins known as MICOS is important for organizing the inner membrane into folds, although exactly how it does so is not fully understood. MICOS consists of at least six different proteins, most of which are found across yeast and animal species. Friedman et al. have now analyzed how the MICOS complex assembles on the inner membrane in yeast cells using a combination of fluorescence and electron microscopy, proteomics and biochemistry. This revealed that in yeast, MICOS is made up of two independent sub-complexes bridged together by a protein called Mic19, which additional experiments suggest controls the number and positions of the cristae junctions that hold the folds of the inner membrane in place.
As part of the approach to understand MICOS complex organization, Friedman et al. removed the six MICOS proteins from yeast cells. Inside these cells, the inner mitochondrial membrane was misfolded. Furthermore, the respiratory complexes did not work normally and as a consequence the cells were unable to grow normally, suggesting that the correct distribution of respiratory complexes in the inner membrane is important for ATP production and depends on MICOS.
These results indicate that MICOS stabilizes the structure of the inner membrane and organizes it into an efficient energy-generating machine. In many human mitochondrial diseases, the inner membrane of mitochondria folds incorrectly, in similar ways to the misfolding seen in the yeast cells that did not contain the MICOS complex. Therefore, the MICOS complex may also influence how these diseases develop.
PMCID: PMC4434539  PMID: 25918844
mitochondria; MICOS; cristae; respiratory complexes; cardiolipin; S. cerevisiae
24.  Comprehensive Evaluation of Toxoplasma gondii VEG and Neospora caninum LIV Genomes with Tachyzoite Stage Transcriptome and Proteome Defines Novel Transcript Features 
PLoS ONE  2015;10(4):e0124473.
Toxoplasma gondii is an important protozoan parasite that infects all warm-blooded animals and causes opportunistic infections in immuno-compromised humans. Its closest relative, Neospora caninum, is an important veterinary pathogen that causes spontaneous abortion in livestock. Comparative genomics of these two closely related coccidians has been of particular interest to identify genes that contribute to varied host cell specificity and disease. Here, we describe a manual evaluation of these genomes based on strand-specific RNA sequencing and shotgun proteomics from the invasive tachyzoite stages of these two parasites. We have corrected predicted structures of over one third of the previously annotated gene models and have annotated untranslated regions (UTRs) in over half of the predicted protein-coding genes. We observe distinctly long UTRs in both the organisms, almost four times longer than other model eukaryotes. We have also identified a putative set of cis-natural antisense transcripts (cis-NATs) and long intergenic non-coding RNAs (lincRNAs). We have significantly improved the annotation quality in these genomes that would serve as a manually curated dataset for Toxoplasma and Neospora research communities.
PMCID: PMC4395442  PMID: 25875305
25.  Comparative genomics reveals insights into avian genome evolution and adaptation 
Zhang, Guojie | Li, Cai | Li, Qiye | Li, Bo | Larkin, Denis M. | Lee, Chul | Storz, Jay F. | Antunes, Agostinho | Greenwold, Matthew J. | Meredith, Robert W. | Ödeen, Anders | Cui, Jie | Zhou, Qi | Xu, Luohao | Pan, Hailin | Wang, Zongji | Jin, Lijun | Zhang, Pei | Hu, Haofu | Yang, Wei | Hu, Jiang | Xiao, Jin | Yang, Zhikai | Liu, Yang | Xie, Qiaolin | Yu, Hao | Lian, Jinmin | Wen, Ping | Zhang, Fang | Li, Hui | Zeng, Yongli | Xiong, Zijun | Liu, Shiping | Zhou, Long | Huang, Zhiyong | An, Na | Wang, Jie | Zheng, Qiumei | Xiong, Yingqi | Wang, Guangbiao | Wang, Bo | Wang, Jingjing | Fan, Yu | da Fonseca, Rute R. | Alfaro-Núñez, Alonzo | Schubert, Mikkel | Orlando, Ludovic | Mourier, Tobias | Howard, Jason T. | Ganapathy, Ganeshkumar | Pfenning, Andreas | Whitney, Osceola | Rivas, Miriam V. | Hara, Erina | Smith, Julia | Farré, Marta | Narayan, Jitendra | Slavov, Gancho | Romanov, Michael N | Borges, Rui | Machado, João Paulo | Khan, Imran | Springer, Mark S. | Gatesy, John | Hoffmann, Federico G. | Opazo, Juan C. | Håstad, Olle | Sawyer, Roger H. | Kim, Heebal | Kim, Kyu-Won | Kim, Hyeon Jeong | Cho, Seoae | Li, Ning | Huang, Yinhua | Bruford, Michael W. | Zhan, Xiangjiang | Dixon, Andrew | Bertelsen, Mads F. | Derryberry, Elizabeth | Warren, Wesley | Wilson, Richard K | Li, Shengbin | Ray, David A. | Green, Richard E. | O’Brien, Stephen J. | Griffin, Darren | Johnson, Warren E. | Haussler, David | Ryder, Oliver A. | Willerslev, Eske | Graves, Gary R. | Alström, Per | Fjeldså, Jon | Mindell, David P. | Edwards, Scott V. | Braun, Edward L. | Rahbek, Carsten | Burt, David W. | Houde, Peter | Zhang, Yong | Yang, Huanming | Wang, Jian | Jarvis, Erich D. | Gilbert, M. Thomas P. | Wang, Jun
Science (New York, N.Y.)  2014;346(6215):1311-1320.
Birds are the most species-rich class of tetrapod vertebrates and have wide relevance across many research fields. We explored bird macroevolution using full genomes from 48 avian species representing all major extant clades. The avian genome is principally characterized by its constrained size, which predominantly arose because of lineage-specific erosion of repetitive elements, large segmental deletions, and gene loss. Avian genomes furthermore show a remarkably high degree of evolutionary stasis at the levels of nucleotide sequence, gene synteny, and chromosomal structure. Despite this pattern of conservation, we detected many non-neutral evolutionary changes in protein-coding genes and noncoding regions. These analyses reveal that pan-avian genomic diversity covaries with adaptations to different lifestyles and convergent evolution of traits.
PMCID: PMC4390078  PMID: 25504712

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