线粒体的基因表达来源于细胞核，但蛋白质合成在线粒体内，它是一组特殊的基因，它与哪些疾病有关呢？本文提供了较全面的综述。
Introduction
The mitochondrial genome encodes a few polypeptides (13 in mammals) that are vital components of the enzyme complexes that perform oxidative phosphorylation (OXPHOS). Since the late 1980s, mutations in mtDNA, including spontaneous deletions and (mainly inherited) point mutations have been recognized as causing a wide spectrum of human diseases associated with bioenergetic defects of mitochondria in the tissues that are most dependent on OXPHOS. These range from mild, late-onset presentations, such as age-related sensorineural-hearing impairment or ocular myopathy (PEO), to devastating and usually fatal infantile disorders, such as Leigh syndrome (also known as fatal necrotizing encephalopathy).
Mitochondrial diseases and nuclear genes
Inherited pathological mutations of mtDNA have generally been considered to be of two classes that either correspond to those in yeast that are classified as mit−, which affects just one of the mtDNA-encoded polypeptides, or syn−, which affects the mitochondrial protein-synthetic apparatus. Those that correspond to yeast syn−, which affect mitochondrial rRNAs or tRNAs, tend to be more pleiotropic in their effects than mit− (although this is not always the case), with patients exhibiting multiple OXPHOS enzyme deficiencies (e.g. complexes I and IV deficiencies, or other combinations). Nuclear mutations affecting individual subunits of the respiratory chain are also well known.
However, there is another category of patients who have multiple OXPHOS enzyme deficiencies, but who do not have any mitochondrial genome mutations. In some of these patients (e.g. in cases of consanguinity), there is clear evidence of autosomal inheritance, and this raises the interesting and important prospect that nuclear genes encoding components of the mitochondrial protein-synthesis machinery might be involved.
Many nuclear genes can be considered as suitable candidates, including those that encode the 80 nuclear proteins of the mitochondrial ribosome [1], in addition to those encoding the mitochondrial aminoacyl-tRNA synthetases, tRNA modification enzymes and translation factors (Figure 1). These constitute, in the main, a dedicated set of genes that is distinct from the homologues that perform similar tasks in cytosolic protein synthesis. Genome sequencing and proteomic approaches have recently succeeded in identifying the vast majority of these candidate genes. However, identifying the crucial genes in which disease-causing mutations are found is a daunting task. Screening 150 or so candidate genes at the DNA sequence level currently remains beyond the capacity of most clinical laboratories. Nevertheless, considerable progress has been made recently, relying either on subtle clues from the molecular phenotype or on high-throughput methods to screen candidate genes.
Figure 1. The composition and provenance of the mitochondrial translational apparatus. rRNAs encoded by the mitochondrial genome are combined with nuclear-coded mitochondrial proteins, generating mitoribosomes. These mitoribosomes then translate the mtDNA-encoded mRNAs using mtDNA-encoded tRNAs. The enzymes for mitochondrial rRNA and tRNA maturation (RNA processing and base-modification) are nuclear-encoded, in addition to all other proteins involved in mitochondrial translation (i.e. aminoacyl-tRNA synthetases, initiation, elongation and termination factors). Mitochondrially encoded transcription products are shown in blue, nuclear-coded proteins in orange. Mitochondrial transcription factor B1 (TFB1M) is also an rRNA methylase.
Pathological mutations affecting the mitoribosome
One example reflects a story we have characterized over the past decade: the tko mutant in Drosophila carries a point mutation in the nuclear gene encoding mitoribosomal protein S12 (MRPS12). The mutation produces a defect in the assembly and/or stability of mitoribosomal small subunits, and an OXPHOS-deficient, mitochondrial disease-like phenotype in the fly [2]. The molecular hallmark of the mutation is a reduced steady-state level of mitochondrial small subunit (12S) compared with large subunit (16S) rRNA. Elpeleg and coworkers noticed a similar defect in fibroblasts from a patient with a fatal, neonatal encephalopathy, born to consanguineous parents who were from a Bedouin family, and decided to investigate the nuclear genes encoding proteins of the mitoribosomal small subunit. Although MRPS12 was found to be intact, they identifed a nonsense mutation in MRPS16, for which the patient was homozygous, almost certainly accounting for the severe defect in mitochondrial-protein synthesis in their cells [3].
In a separate study, the groups of Smeitink and Shoubridge have been investigating two siblings with a slightly different phenotype that includes postnatal liver failure and has a generalized deficiency of mitochondrial-protein synthesis in patient-derived fibroblasts. In this case, they identified, by means of chromosome transfer, microsatellite and sequence analysis, and complementation in cell culture [4], a mis-sense mutation in EFG1, one of two genes encoding different isoforms of the mitochondrial translational elongation factor G [5]. However, the exact role of the different mitochondrial EF-G homologues is unclear. Perhaps they perform similar tasks in different tissues or they operate under different physiological conditions. The fact that the duplication is ancient (i.e. common to all eukaryotes), suggests that they have functionally distinct roles in the ribosome cycle. The yeast homologue of EFG1 (but not of EFG2) is functionally essential for OXPHOS; therefore, it has been suggested that EFG2 has an auxiliary role.
In the family studied by the Smeitink and Shoubridge groups, the point mutation (A521G) affects a conserved residue of the GTP-binding site of EFG1, and is predicted to have a drastic effect on nucleotide binding and hence enzymatic function. However, mitochondrial-protein synthesis in patient-derived fibroblasts was not completely abolished, indicating significant residual activity, possibly provided, at least in part, by EFG2. Transfection of a cDNA encoding EFG1, but not one encoding EFG2, led to phenotypic complementation. It will be interesting to determine whether knockdown of EFG2 conversely exacerbates the defect.
Pathological mutations in other genes influencing mitochondrial translation
Another class of mitochondrial-protein-synthesis defects that has recently come to light involves tRNA base-modification. Base modification at the wobble base seems to be important for efficient and accurate decoding and is the molecular ‘target’ of several pathological tRNA gene mutations in mtDNA itself 6 and 7. In general, mitochondrial tRNAs deviate from the canonical structure, and several types of base modifications (e.g. pseudouridylation) might be important for maintaining the proper tertiary structure. In yeast, a family of pseudourine synthases with complex and multiple specificities is involved in performing this isomerization at diverse positions of cytosolic and mitochondrial tRNAs and rRNAs. The human gene pseudouridylate synthase 1 (PUS1), which encodes one such isoenzyme, has recently been shown to harbour a mutation in a patient with mitochondrial myopathy and sideroblastic anemia [8], associated with defective OXPHOS in bone marrow and skeletal muscle. Although a direct effect on mitochondrial-protein synthesis has not yet been demonstrated, deficient pseudouridylation of one or more mitochondrial tRNAs seems to be the most plausible disease mechanism. Nuclear genes encoding enzymes for base-modification of mitochondrial tRNAs are also strong candidates as phenotypic modifiers of the mtDNA A1555G mutation [9].
In yeast, a diverse set of mitochondrial translation factors, whose roles seem to be specific to individual mRNAs, has been known about for more than a decade. In general, these factors interact either with the 5′ untranslated regions (5′UTRs) of specific mitochondrial mRNAs or with other proteins that do so, and form a complex that is both essential for efficient translation and involved in the coordination of assembly of the OXPHOS complex to which the polypeptide product is destined [10]. This link between translation and assembly is probably dictated by the extremely hydrophobic nature of many of the mitochondrial translation products. The existence of comparable factors in mammalian mitochondria has long been a subject of speculation. On the one hand, mammalian mitochondrial mRNAs lack 5′UTRs entirely. On the other hand, most aspects of OXPHOS biogenesis are functionally conserved phylogenetically, leading to the suggestion that such factors, if they exist, interact with the coding regions of mammalian mitochondrial mRNAs. One problem with this idea has always been that the relevant factors are poorly conserved at the primary sequence level even between different fungi, making their counterparts difficult to identify in the genomes of organisms belonging to other kingdoms, such as metazoans.
A potential breakthrough in this area has recently been made by the groups of Robinson and Lander 11 and 12, studying the gene encoding the leucine-rich pentatricopeptide repeat cassette (LRPPRC), which is involved in the French Canadian variant of COX-deficient Leigh syndrome (LSFC). LRPPRC encodes an RNA-binding protein that is a distant homologue of the yeast (Saccharomyces cerevisiae) protein Pet309p, a factor that is specific for the translation and stabilization of the mitochondrially encoded COX1 mRNA [13]. The human LRPPRC protein is also found in mitochondria, although some reports indicate that it might also be present in the nucleus or endoplasmic reticulum (ER). An LSFC mutation in LRPPRC, A354V, results in greatly reduced expression of the protein inside mitochondria and lower levels of both COXI and COXIII mRNAs [12], accompanied by the synthesis of a novel and as yet unidentified mitochondrial translation product. These findings suggest that LRPPRC has an analogous role in human mitochondria to that played by Pet309p in yeast. The pentatrichopeptide repeat (PPR) superfamily appears to have expanded massively in the genomes of land plants, where its members are believed to have diverse roles in organellar RNA metabolism.
Concluding remarks
If the hypothesis concerning LRPPRC proves to be correct, we can expect that distant homologues of other translational regulators in yeast mitochondria will emerge as human disease genes in other contexts. In addition, we can expect an analogous class of such genes serving as translational regulators and assembly cofactors for mtDNA-encoded complex I subunits. Furthermore, the genes encoding factors of this class might also be good candidates for the hypothesized, but still largely elusive, genetic modifiers of mtDNA disease phenotype.
Acknowledgements
We thank Orly Elpeleg and Jan Smeitink for communicating their findings to us ahead of publication. Our own research is supported by the Academy of Finland, Tampere University Medical Research Fund, The Wellcome Trust, Muscular Dystrophy Campaign and The European union (MitEURO and EUMITOCOMBAT projects).
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