The process of mammalian mitochondrial protein synthesis Mai, Nicole; Chrzanowska-Lightowlers, Zofia M. A.; Lightowlers, Robert N.
Cell & tissue research/Cell and tissue research,
01/2017, Letnik:
367, Številka:
1
Journal Article
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Oxidative phosphorylation (OXPHOS) is the mechanism whereby ATP, the major energy source for the cell, is produced by harnessing cellular respiration in the mitochondrion. This is facilitated by five ...multi-subunit complexes housed within the inner mitochondrial membrane. These complexes, with the exception of complex II, are of a dual genetic origin, requiring expression from nuclear and mitochondrial genes. Mitochondrially encoded mRNA is translated on the mitochondrial ribosome (mitoribosome) and the recent release of the near atomic resolution structure of the mammalian mitoribosome has highlighted its peculiar features. However, whereas some aspects of mitochondrial translation are understood, much is to be learnt about the presentation of mitochondrial mRNA to the mitoribosome, the biogenesis of the machinery, the exact role of the membrane, the constitution of the translocon/insertion machinery and the regulation of translation in the mitochondrion. This review addresses our current knowledge of mammalian mitochondrial gene expression, highlights key questions and indicates how defects in this process can result in profound mitochondrial disease.
Transplantation of functional mitochondria directly into defective cells is a novel approach that has recently caught the attention of scientists and the general public alike. Could this be too good ...to be true?
The bacterial Ras-like protein Era has been reported previously to bind 16S rRNA within the 30S ribosomal subunit and to play a crucial role in ribosome assembly. An orthologue of this essential ...GTPase ERAL1 (Era G-protein-like 1) exists in higher eukaryotes and although its exact molecular function and cellular localization is unknown, its absence has been linked to apoptosis. In the present study we show that human ERAL1 is a mitochondrial protein important for the formation of the 28S small mitoribosomal subunit. We also show that ERAL1 binds in vivo to the rRNA component of the small subunit 12S mt (mitochondrial)-rRNA. Bacterial Era associates with a 3' unstructured nonanucleotide immediately downstream of the terminal stem-loop (helix 45) of 16S rRNA. This site contains an AUCA sequence highly conserved across all domains of life, immediately upstream of the anti-Shine-Dalgarno sequence, which is conserved in bacteria. Strikingly, this entire region is absent from 12S mt-rRNA. We have mapped the ERAL1-binding site to a 33 nucleotide section delineating the 3' terminal stem-loop region of 12S mt-rRNA. This loop contains two adenine residues that are reported to be dimethylated on mitoribosome maturation. Furthermore, and also in contrast with the bacterial orthologue, loss of ERAL1 leads to rapid decay of nascent 12S mt-rRNA, consistent with a role as a mitochondrial RNA chaperone. Finally, whereas depletion of ERAL1 leads to apoptosis, cell death occurs prior to any appreciable loss of mitochondrial protein synthesis or reduction in the stability of mitochondrial mRNA.
Abstract
LonP1 is a mitochondrial matrix protease whose selective substrate specificity is essential for maintaining mitochondrial homeostasis. Recessively inherited, pathogenic defects in LonP1 have ...been previously reported to underlie cerebral, ocular, dental, auricular and skeletal anomalies (CODAS) syndrome, a complex multisystemic and developmental disorder. Intriguingly, although classical mitochondrial disease presentations are well-known to exhibit marked clinical heterogeneity, the skeletal and dental features associated with CODAS syndrome are pathognomonic. We have applied whole exome sequencing to a patient with congenital lactic acidosis, muscle weakness, profound deficiencies in mitochondrial oxidative phosphorylation associated with loss of mtDNA copy number and MRI abnormalities consistent with Leigh syndrome, identifying biallelic variants in the LONP1 (NM_004793.3) gene; c.1693T > C predicting p.(Tyr565His) and c.2197G > A predicting p.(Glu733Lys); no evidence of the classical skeletal or dental defects observed in CODAS syndrome patients were noted in our patient. In vitro experiments confirmed the p.(Tyr565His) LonP1 mutant alone could not bind or degrade a substrate, consistent with the predicted function of Tyr565, whilst a second missense p.(Glu733Lys) variant had minimal effect. Mixtures of p.(Tyr565His) mutant and wild-type LonP1 retained partial protease activity but this was severely depleted when the p.(Tyr565His) mutant was mixed with the p.(Glu733Lys) mutant, data consistent with the compound heterozygosity detected in our patient. In summary, we conclude that pathogenic LONP1 variants can lead to a classical mitochondrial disease presentations associated with severe biochemical defects in oxidative phosphorylation in clinically relevant tissues.
Human mitochondria contain their own genome, mitochondrial DNA, that is expressed in the mitochondrial matrix. This genome encodes 13 vital polypeptides that are components of the multisubunit ...complexes that couple oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane that houses these complexes comprises the inner boundary membrane that runs parallel to the outer membrane, infoldings that form the cristae membranes, and the cristae junctions that separate the two. It is in these cristae membranes that the OXPHOS complexes have been shown to reside in various species. The majority of the OXPHOS subunits are nuclear-encoded and must therefore be imported from the cytosol through the outer membrane at contact sites with the inner boundary membrane. As the mitochondrially encoded components are also integral members of these complexes, where does protein synthesis occur? As transcription, mRNA processing, maturation, and at least part of the mitoribosome assembly process occur at the nucleoid and the spatially juxtaposed mitochondrial RNA granules, is protein synthesis also performed at the RNA granules close to these entities, or does it occur distal to these sites? We have adapted a click chemistry-based method coupled with stimulated emission depletion nanoscopy to address these questions. We report that, in human cells in culture, within the limits of our methodology, the majority of mitochondrial protein synthesis is detected at the cristae membranes and is spatially separated from the sites of RNA processing and maturation.
The Escherichia coli oligoribonuclease, ORN, has a 3' to 5' exonuclease activity specific for small oligomers that is essential for cell viability. The human homologue, REXO2, has hitherto been ...incompletely characterized, with only its in vitro ability to degrade small single-stranded RNA and DNA fragments documented. Here we show that the human enzyme has clear dual cellular localization being present both in cytosolic and mitochondrial fractions. Interestingly, the mitochondrial form localizes to both the intermembrane space and the matrix. Depletion of REXO2 by RNA interference causes a strong morphological phenotype in human cells, which show a disorganized network of punctate and granular mitochondria. Lack of REXO2 protein also causes a substantial decrease of mitochondrial nucleic acid content and impaired de novo mitochondrial protein synthesis. Our data constitute the first in vivo evidence for an oligoribonuclease activity in human mitochondria.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mitochondrial diseases are clinically heterogeneous disorders caused by a wide spectrum of mutations in genes encoded by either the nuclear or the mitochondrial genome. Treatments for mitochondrial ...diseases are currently focused on symptomatic management rather than improving the biochemical defect caused by a particular mutation. This review focuses on the latest advances in the development of treatments for mitochondrial disease, both small molecules and gene therapies, as well as methods to prevent transmission of mitochondrial disease through the germline.
Russell et al. review recent progress in the varied approaches to treat and prevent mitochondrial disease, including small-molecule-based approaches to improve mitochondrial function or turnover, manipulation of the mitochondrial genome, and mitochondrial replacement therapy.
Various specialized domains have been described in the cytosol and the nucleus; however, little is known about compartmentalization within the mitochondrial matrix. GRSF1 (G-rich sequence factor 1) ...is an RNA binding protein that was previously reported to localize in the cytosol. We found that an isoform of GRSF1 accumulates in discrete foci in the mitochondrial matrix. These foci are composed of nascent mitochondrial RNA and also contain RNase P, an enzyme that participates in mitochondrial RNA processing. GRSF1 was found to interact with RNase P and to be required for processing of both classical and tRNA-less RNA precursors. In its absence, cleavage of primary RNA transcripts is abnormal, leading to decreased expression of mitochondrially encoded proteins and mitochondrial dysfunction. Our findings suggest that the foci containing GRSF1 and RNase P correspond to sites where primary RNA transcripts converge to be processed. We have termed these large ribonucleoprotein structures “mitochondrial RNA granules.”
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► GRSF1 resides in the mitochondrial matrix and is required for mitochondrial function ► GRSF1 is required for the processing of tRNA-containing and tRNA-lacking precursors ► GRSF1, RNase P, and nascent RNA are part of “mitochondrial RNA granules” ► Mitochondrial RNA granules are functionally linked to RNA processing
Mitochondrial diseases are among the most common and most complex of all inherited genetic diseases. The involvement of both the mitochondrial and nuclear genome presents unique challenges, but ...despite this there have been some remarkable advances in our knowledge of mitochondrial diseases over the past few years. A greater understanding of mitochondrial genetics has led to improved diagnosis as well as novel ways to prevent transmission of severe mitochondrial disease. These and other advances have had a major impact on patient care, but considerable challenges remain, particularly in the areas of therapies for those patients manifesting clinical symptoms associated with mitochondrial dysfunction and the tissue specificity seen in many mitochondrial disorders. This review highlights some important recent advances in mitochondrial disease but also stresses the areas where progress is essential.
OXA1, the mitochondrial member of the YidC/Alb3/Oxa1 membrane protein insertase family, is required for the assembly of oxidative phosphorylation complexes IV and V in yeast. However, depletion of ...human OXA1 (OXA1L) was previously reported to impair assembly of complexes I and V only. We report a patient presenting with severe encephalopathy, hypotonia and developmental delay who died at 5 years showing complex IV deficiency in skeletal muscle. Whole exome sequencing identified biallelic OXA1L variants (c.500_507dup, p.(Ser170Glnfs*18) and c.620G>T, p.(Cys207Phe)) that segregated with disease. Patient muscle and fibroblasts showed decreased OXA1L and subunits of complexes IV and V. Crucially, expression of wild‐type human OXA1L in patient fibroblasts rescued the complex IV and V defects. Targeted depletion of OXA1L in human cells or Drosophila melanogaster caused defects in the assembly of complexes I, IV and V, consistent with patient data. Immunoprecipitation of OXA1L revealed the enrichment of mtDNA‐encoded subunits of complexes I, IV and V. Our data verify the pathogenicity of these OXA1L variants and demonstrate that OXA1L is required for the assembly of multiple respiratory chain complexes.
Synopsis
This work describes the first confirmed pathogenic variants in OXA1L in a mitochondrial disease patient with tissue‐specific combined OXPHOS deficiencies. OXA1L was shown to be important for complex IV assembly in human cell lines with targeted depletion of OXA1L and in patient tissues.
Bi‐allelic variants in OXA1L (NM_005015.3; c.500_507dup, p.(Ser170Glnfs*18); c.620G>T, p.(Cys207Phe) were identified by whole exome sequencing in a patient with hypotonia, developmental delay and severe encephalopathy who died following cardiorespiratory arrest.
Patient fibroblasts showed decreased protein levels of OXA1L and subunits of OXPHOS complexes I, IV and V.
Introduction of wild‐type OXA1L into patient fibroblasts by retroviral transduction rescued the OXPHOS defects, confirming pathogenicity of the OXA1L variants.
Depletion of OXA1L by siRNA in human cell lines and Drosophila caused combined OXPHOS deficiencies including severe effects on complex IV.
An OXA1L CRISPR/Cas9 knockout cell line with inducible expression of FLAG‐tagged OXA1L was used for OXA1L immunoprecipitation and identified an enrichment of mtDNA‐encoded subunits of complexes I, IV and V.
This work describes the first confirmed pathogenic variants in OXA1L in a mitochondrial disease patient with tissue specific combined OXPHOS deficiencies. OXA1L was shown to be important for complex IV assembly in human cell lines with targeted depletion of OXA1L and in patient tissues.
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