Mapping gene associations in human mitochondria using clinical disease phenotypes

Nuclear genes encode most mitochondrial proteins, and their mutations cause diverse and debilitating clinical disorders. To date, 1,200 of these mitochondrial genes have been recorded, while no standardized catalog exists of the associated clinical phenotypes. Such a catalog would be useful to develop methods to analyze human phenotypic data, to determine genotype-phenotype relations among many genes and diseases, and to support the clinical diagnosis of mitochondrial disorders. Here we establish a clinical phenotype catalog of 174 mitochondrial disease genes and study associations of diseases and genes. Phenotypic features such as clinical signs and symptoms were manually annotated from full-text medical articles and classified based on the hierarchical MeSH ontology. This classification of phenotypic features of each gene allowed for the comparison of diseases between different genes. In turn, we were then able to measure the phenotypic associations of disease genes for which we calculated a quantitative value that is based on their shared phenotypic features. The results showed that genes sharing more similar phenotypes have a stronger tendency for functional interactions, proving the usefulness of phenotype similarity values in disease gene network analysis. We then constructed a functional network of mitochondrial genes and discovered a higher connectivity for non-disease than for disease genes, and a tendency of disease genes to interact with each other. Utilizing these differences, we propose 168 candidate genes that resemble the characteristic interaction patterns of mitochondrial disease genes. Through their network associations, the candidates are further prioritized for the study of specific disorders such as optic neuropathies and Parkinson disease. Most mitochondrial disease phenotypes involve several clinical categories including neurologic, metabolic, and gastrointestinal disorders, which might indicate the effects of gene defects within the mitochondrial system. The accompanying knowledgebase (http://www.mitophenome.org/) supports the study of clinical diseases and associated genes

Advances in Human Mitochondria-Based Therapies

Mitochondria are the key biological generators of eukaryotic cells, controlling the energy supply while providing many important biosynthetic intermediates. Mitochondria act as a dynamic, functionally and structurally interconnected network hub closely integrated with other cellular compartments via biomembrane systems, transmitting biological information by shuttling between cells and tissues. Defects and dysregulation of mitochondrial functions are critically involved in pathological mechanisms contributing to aging, cancer, inflammation, neurodegenerative diseases, and other severe human diseases. Mediating and rejuvenating the mitochondria may therefore be of significant benefit to prevent, reverse, and even treat such pathological conditions in patients. The goal of this review is to present the most advanced strategies using mitochondria to manage such disorders and to further explore innovative approaches in the field of human mitochondriabased therapies

Replication-Transciption Switch in Human Mitochondria

Coordinated replication and expression of mitochondrial genome is critical for metabolically active cells during various stages of development. However, it is not known whether replication and transcription can occur simultaneously without interfering with each other and whether mtDNA copy number can be regulated by the transcription machinery. Human mitochondrial RNA polymerase (mtRNAP) is a central enzyme involved in gene expression in mitochondria. It generates genome-size polycistronic transcripts and also makes replication primers at two origins of replication. MtRNAP is distantly related to phage T7 RNAP. While T7 RNAP is optimized to produce large amounts of transcripts to overcompete the bacterial RNAP, mtRNAP must coordinate RNA synthesis with processing and translation. We hypothesized that mtRNAP must be slower than T7 RNAP and measured elongation rates for these RNAPs. We found that mtRNAP is about 20 times slower than T7 RNAP. We also found that mtRNAP is inherently non-processive and cannot synthesize long transcripts. We proposed that low processivity and slow elongation rates of mtRNAP requires assistance of an additional elongation factor. We show that interaction of a recently identified human transcription elongation factor, TEFM, with mtRNAP dramatically increases processivity and elongation rates of the mitochondrial transcription machinery. Importantly, we found that TEFM prevents premature transcription termination and thus generation of replication primers by mtRNAP. Thus, TEFM serves as a component of a molecular switch between replication and transcription, which appear to be mutually exclusive processes in mitochondria. The switch likely allows avoiding the detrimental consequences of head-on collisions between replication and transcription machineries. Regulation of TEFM may explain how mtRNAP transcription rates and, as consequence, respiration and ATP production, can be increased in mitochondria without the need to replicate mtDNA, which has been observed during different developmental processes

Localisation of the human hSuv3p helicase in the mitochondrial matrix and its preferential unwinding of dsDNA

We characterised the human hSuv3p protein belonging to the family of NTPases/helicases. In yeast mitochondria the hSUV3 orthologue is a component of the degradosome complex and participates in mtRNA turnover and processing, while in Caenorhabditis elegans the hSUV3 orthologue is necessary for viability of early embryos. Using immunofluorescence analysis, an in vitro mitochondrial uptake assay and sub‐fractionation of human mitochondria we show hSuv3p to be a soluble protein localised in the mitochondrial matrix. We expressed and purified recombinant hSuv3p protein from a bacterial expression system. The purified enzyme was capable of hydrolysing ATP with a Km of 41.9 µM and the activity was only modestly stimulated by polynucleotides. hSuv3p unwound partly hybridised dsRNA and dsDNA structures with a very strong preference for the latter. The presented analysis of the hSuv3p NTPase/helicase suggests that new functions of the protein have been acquired in the course of evolution

Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns

A highly efficient RNA-synthesizing system with isolated HeLa cell mitochondria has been developed and characterized regarding its requirements and its products. In this system, transcription is initiated and the transcripts are processed in a way which closely reproduces the in vivo patterns. Total RNA labeling in isolated mitochondria proceeds at a constant rate for about 30 min at 37 degrees C; the estimated rate of synthesis is at least 10 to 15% of the in vivo rate. Polyadenylation of the mRNAs is less extensive in this system than in vivo. Furthermore, compared with the in vivo situation, rRNA synthesis in vitro is less efficient than mRNA synthesis. This is apparently due to a decreased rate of transcription initiation at the rRNA promoter and probably a tendency also for premature termination of the nascent rRNA chains. The 5'-end processing of rRNA also appears to be slowed down, and it is very sensitive to the incubation conditions, in contrast to mRNA processing. It is suggested that the lower efficiency and the lability of rRNA synthesis and processing in isolated mitochondria may be due to cessation of import from the cytoplasm of ribosomal proteins that play a crucial role in these processes. The formation of the light-strand-coded RNA 18 (7S RNA) is affected by high pH or high ATP concentration differently from the overall light-strand transcription. The dissociation of the two processes may have important implications for the mechanism of formation and the functional role of this unusual RNA species. The high efficiency, initiation capacity, and processing fidelity of the in vitro RNA-synthesizing system described here make it a valuable tool for the analysis of the role of nucleocytoplasmic-mitochondrial interactions in organelle gene expression

PDE12 removes mitochondrial RNA poly(A) tails and controls translation in human mitochondria

Polyadenylation of mRNA in human mitochondria is crucial for gene expression and perturbation of poly(A) tail length has been linked to a human neurodegenerative disease. Here we show that 2′-phosphodiesterase (2′-PDE), (hereafter PDE12), is a mitochondrial protein that specifically removes poly(A) extensions from mitochondrial mRNAs both in vitro and in mitochondria of cultured cells. In eukaryotes, poly(A) tails generally stabilize mature mRNAs, whereas in bacteria they increase mRNA turnover. In human mitochondria, the effects of increased PDE12 expression were transcript dependent. An excess of PDE12 led to an increase in the level of three mt-mRNAs (ND1, ND2 and CytB) and two (CO1 and CO2) were less abundant than in mitochondria of control cells and there was no appreciable effect on the steady-state level of the remainder of the mitochondrial transcripts. The alterations in poly(A) tail length accompanying elevated PDE12 expression were associated with severe inhibition of mitochondrial protein synthesis, and consequently respiratory incompetence. Therefore, we propose that mRNA poly(A) tails are important in regulating protein synthesis in human mitochondria, as it is the case for nuclear-encoded eukaryotic mRNA

Human mitochondrial degradosome prevents harmful mitochondrial R loops and mitochondrial genome instability

R loops are nucleic acid structures comprising an DNA-RNA hybrid and a displaced single-stranded DNA. These structures may occur transiently during transcription, playing essential biological functions. However, persistent R loops may become pathological as they are important drivers of genome instability and have been associated with human diseases. The mitochondrial degradosome is a functionally conserved complex from bacteria to human mitochondria. It is composed of the ATP-dependent RNA and DNA helicase SUV3 and the PNPase ribonuclease, playing a central role in mitochondrial RNA surveillance and degradation. Here we describe a new role for the mitochondrial degradosome in preventing the accumulation of pathological R loops in the mitochondrial DNA, in addition to preventing dsRNA accumulation. Our data indicate that, similar to the molecular mechanisms acting in the nucleus, RNA surveillance mechanisms in the mitochondria are crucial to maintain its genome integrity by counteracting pathological R-loop accumulation.European Research Council ERC2014 AdG669898 TARLOOPMinisterio de Economía y Competitividad BFU2013-42918-P, BFU2016-75058-

Pre-microRNA and Mature microRNA in Human Mitochondria

Chantier qualité GAInternational audienceBACKGROUND: Because of the central functions of the mitochondria in providing metabolic energy and initiating apoptosis on one hand and the role that microRNA (miRNA) play in gene expression, we hypothesized that some miRNA could be present in the mitochondria for post-transcriptomic regulation by RNA interference. We intend to identify miRNA localized in the mitochondria isolated from human skeletal primary muscular cells. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the potential origin of mitochondrial miRNA, we in-silico searched for microRNA candidates in the mtDNA. Twenty five human pre-miRNA and 33 miRNA aligments (E-value35) for the smallest RNA input concentration and 204 miRNA for the maximum RNA input concentration. In silico analysis predicted 80 putative miRNA target sites in the mitochondrial genome (E-value<0.05). CONCLUSIONS/SIGNIFICANCE: The present study experimentally demonstrated for the first time the presence of pre-miRNA and miRNA in the human mitochondria isolated from skeletal muscular cells. A set of miRNA were significantly detected in mitochondria fraction. The origin of these pre-miRNA and miRNA should be further investigate to determine if they are imported from the cytosol and/or if they are partially processed in the mitochondria

Mechanism of Transcription Anti-termination in Human Mitochondria.

In human mitochondria, transcription termination events at a G-quadruplex region near the replication origin are thought to drive replication of mtDNA by generation of an RNA primer. This process is suppressed by a key regulator of mtDNA-the transcription factor TEFM. We determined the structure of an anti-termination complex in which TEFM is bound to transcribing mtRNAP. The structure reveals interactions of the dimeric pseudonuclease core of TEFM with mobile structural elements in mtRNAP and the nucleic acid components of the elongation complex (EC). Binding of TEFM to the DNA forms a downstream sliding clamp, providing high processivity to the EC. TEFM also binds near the RNA exit channel to prevent formation of the RNA G-quadruplex structure required for termination and thus synthesis of the replication primer. Our data provide insights into target specificity of TEFM and mechanisms by which it regulates the switch between transcription and replication of mtDNA

Markedly different ATP requirements for rRNA synthesis and mtDNA light strand transcription versus mRNA synthesis in isolated human mitochondria

In isolated mitochondria from HeLa cells, the ATP requirements for mitochondral DNA (mtDNA) transcription and RNA processing can be satisfied by either endogenous synthesis, mainly through oxidative- phosphorylation, or by exogenous supply. The pattern of RNA synthesis changes dramatically depending upon the level of ATP available. At the low intramitochondrial ATP levels produced from endogenous ADP in the presence of an oxidizable substrate and phosphate, the mRNA species are labeled to a substantial extent, whereas there is only a marginal labeling of the rRNA species and light (L) strand transcripts. By contrast, high ATP levels, either provided exogenously or produced endogenously in the presence of an oxidizable substrate, phosphate, and exogenous ADP, strongly stimulate rRNA synthesis (about 10-fold) and light (L) strand transcription (greater than 10-fold), with only a slight increase in mRNA synthesis