How do mammalian mitochondria synthesize proteins?

Mitochondria contain their own genome that is expressed by nuclear-encoded factors imported into the organelle. This review provides a summary of the current state of knowledge regarding the mechanism of protein translation in human mitochondria and the factors involved in this process.

Isolation and identification of a protein binding to the localization element of Metallothionein-1 mRNA.

mRNA localization provides a mechanism for localized protein synthesis. mRNAs encoding certain proteins, including c-MYC, c-FOS, MT-1 (Metallothionein-1) and vimentin, are localized around the nuclei of mammalian cells and are associated with the cytoskeleton. Targeting of these mRNAs to the perinuclear cytoplasm is mediated by elements within their 3'-UTRs (3'-untranslated regions), but many of the trans-acting proteins remain unidentified. UV cross-linking assays using radiolabelled transcripts indicated that a protein of approx. 50 kDa (from the Chinese-hamster ovary cell extracts) bound to the MT-1 3'-UTR sequence. Competition experiments using unlabelled mutant 3'-UTR RNAs revealed that the binding of this protein is specific to localization-positive mutants. Isolation of a 50 kDa protein was achieved by an RNA affinity-based method in which biotinylated MT-1 3'-UTR RNA was anchored to paramagnetic beads. Bound proteins were eluted and analysed by SDS/PAGE. The 50 kDa protein was extracted from the gel, subjected to trypsin digestion and identified by matrix-assisted laser-desorption/ionization-time-of-flight mass spectrometry as eukaryote elongation factor 1alpha.

Sodium channel mRNAs at the neuromuscular junction: distinct patterns of accumulation and effects of muscle activity.

Voltage-gated sodium channels (VGSCs) are highly concentrated at the neuromuscular junction (NMJ) in mammalian skeletal muscle. Here we test the hypothesis that local upregulation of mRNA contributes to this accumulation. We designed radiolabeled antisense RNA probes, specific for the "adult" Na(V)1.4 and "fetal" Na(V)1.5 isoforms of VGSC in mammalian skeletal muscle, and used them in in situ hybridization studies of rat soleus muscles. Na(V)1.4 mRNA is present throughout normal adult muscles but is highly concentrated at the NMJ, in which the amount per myonucleus is more than eightfold greater than away from the NMJ. Na(V)1.5 mRNA is undetectable in innervated muscles but is dramatically upregulated by denervation. In muscles denervated for 1 week, both Na(V)1.4 and Na(V)1.5 mRNAs are present throughout the muscle, and both are concentrated at the NMJ. No Na(V)1.5 mRNA was detectable in denervated muscles stimulated electrically for 1 week in vivo. Neither denervation nor stimulation had any significant effect on the level or distribution of Na(V)1.4 mRNA. We conclude that factors, probably derived from the nerve, lead to the increased concentration of VGSC mRNAs at the NMJ. In addition, the expression of Na(V)1.5 mRNA is downregulated by muscle activity, both at the NMJ and away from it.

Absence of expression from RNA internalised into electroporated mammalian mitochondria.

Transfection of mammalian mitochondria has proved to be notoriously difficult. Whilst there have been sporadic reports of import of foreign nucleic acids into isolated organelles, these imported nucleic acids have never been demonstrated to be functional. Inability to manipulate mitochondrial gene expression has hampered our understanding of RNA processing, maturation and translation in mitochondria. In an attempt to establish a model system for mt-RNA expression, we have electroporated rat liver mitochondria and mitoplasts in the presence of various RNA constructs built around the mitochondrial reporter gene mt-luciferase. Following electroporation, a fraction of the RNA was shown to be stably maintained, mitochondria remained coupled for oxidative phosphorylation and intramitochondrial protein synthesis was unaffected. In no case, however, was this RNA translated.

Fending off decay: a combinatorial approach in intact cells for identifying mRNA stability elements.

The strategy of systematic evolution, whereby nucleic acid sequences or conformers can be selected and amplified from a randomized population, has been exploited by many research groups for numerous purposes. It is, however, a technique largely performed in vitro, under nonphysiological conditions. We have now modified this in vitro approach to accomplish selection in growing cells. Here, we report that this new methodology has been used in vivo to select RNA elements that confer increased transcript stability. A randomized cassette was embedded in a 3'-untranslated region (UTR), downstream from the luciferase reporter open reading frame. A heterogeneous population of capped luciferase mRNA was then generated by in vitro transcription. Human liver Hep G2 cells were electroporated with this population of luciferase mRNA and total cytoplasmic RNA was isolated after varying lengths of incubation. Following RT-PCR, the 3' UTR was used to reconstruct a new population of luciferase templates, permitting subsequent cycles of in vitro transcription, electroporation, RNA isolation, and RT-PCR. Increasing the incubation time at each cycle before RNA isolation imposed selection for stable transcripts. The functional half-life of the luciferase mRNA population increased from 55 to 140 min after four cycles. Subsequent sequencing of the selected 3' UTRs revealed G-U rich elements in clones with extended chemical and functional half-lives.

Inhibition of mitochondrial protein synthesis promotes autonomous regulation of mtDNA expression and generation of a new mitochondrial RNA species.

Mammalian mitochondria are known to proliferate in response to several stimuli. Proliferation requires an increase in expression of genes encoding proteins involved in mitochondrial biogenesis, as well as in the replication and expression of mitochondrial DNA (mtDNA). In contrast, we report that inhibiting mitochondrial protein synthesis causes a modulation in mtDNA gene expression without the concomitant increase in proliferative markers. Further, inhibition results in the production of a previously unidentified light-strand mitochondrial RNA that spans the entire displacement loop, the function of which is currently unknown.

The mouse tumor cell lines EL4 and RMA display mosaic expression of NK-related and certain other surface molecules and appear to have a common origin.

As a potential means for facilitating studies of NK cell-related molecules, we examined the expression of these molecules on a range of mouse tumor cell lines. Of the lines we initially examined, only EL4 and RMA expressed such molecules, both lines expressing several members of the Ly49 and NKRP1 families. Unexpectedly, several of the NK-related molecules, together with certain other molecules including CD2, CD3, CD4, CD32, and CD44, were often expressed in a mosaic manner, even on freshly derived clones, indicating frequent switching in expression. In each case examined, switching was controlled at the mRNA level, with expression of CD3zeta determining expression of the entire CD3-TCR complex. Each of the variable molecules was expressed independently, with the exception that CD3 was restricted to cells that also expressed CD2. Treatment with drugs that affect DNA methylation and histone acetylation could augment the expression of at least some of the variable molecules. The striking phenotypic similarity between EL4 and RMA led us to examine the state of their TCRbeta genes. Both lines had identical rearrangements on both chromosomes, indicating that RMA is in fact a subline of EL4. Overall, these findings suggest that EL4 is an NK-T cell tumor that may have retained a genetic mechanism that permits the variable expression of a restricted group of molecules involved in recognition and signaling.

Does the mitochondrial transcription-termination complex play an essential role in controlling differential transcription of the mitochondrial DNA?

The mechanism of mitochondrial transcription is well documented although the method of regulation remains obscure. The mitochondrial transcription-termination complex, mTERF, holds a key position in determining the fate of heavy-strand promotor-initiated transcripts and has been suggested as a candidate in the regulation of mitochondrial DNA (mtDNA) transcription. We report here the first example of a modulation of mTERF-complex binding activity concomitant with a differential mtDNA transcription rate. We suggest that these observations are indicative of a method of intra-organellar transcriptional fine tuning.

Stochastic acquisition of Qa1 receptors during the development of fetal NK cells in vitro accounts in part but not in whole for the ability of these cells to distinguish between class I-sufficient and class I-deficient targets.

Fetal mouse NK cells are grossly deficient in the expression of Ly49 molecules yet show a limited ability to distinguish between wild-type and MHC class I-deficient target cells. In this paper we report that during their development in vitro from immature thymic progenitors, a proportion of C57BL/6 fetal NK cells acquires receptors for a soluble form of the nonclassical class I molecule Qa1b associated with the Qdm peptide, but not for soluble forms of the classical class I molecules Kb and Db. The acquisition of these Qa1 receptors occurs in a stochastic manner that is strictly controlled by cytokines, and in particular is strongly inhibited by IL-4. All fetal NK clones tested, including those that lack detectable Qa1 receptors, express mRNA for CD94 and for both inhibitory and noninhibitory members of the NKG2 family. Fetal NK cells lacking receptors for Qa1 (and also for classical class I molecules) cannot distinguish between wild-type and class I-deficient blasts but, surprisingly, distinguish efficiently between certain wild-type and class I-deficient tumor cells. A variant line that lacks several members of the NKG2 family kills both types of tumor cell equally well, suggesting the existence of NKG2-containing inhibitory receptors that recognize as yet undefined nonclassical class I molecules of restricted distribution.

Conversion of a reporter gene for mitochondrial gene expression using iterative mega-prime PCR.

Mammalian mitochondria possess their own multicopy genome, mtDNA. Although much is known about mtDNA replication and transcription, our knowledge of the mechanisms governing mt-RNA processing, stability and translation remains rudimentary. We have taken a step towards addressing these issues by altering the luciferase reporter gene to accommodate the variation in mitochondrial codon recognition. 19 essential substitutions have been generated by an iterative mega-primer PCR technique. To mimic mt-mRNA species and to optimise intramitochondrial translation, further engineering has produced a template which, when transcribed in vitro, generates an RNA species with only two nucleotides upstream from the initiation codon, an absence of a 3' untranslated region and a polyadenylated tail of 40 residues. It is intended that mt-luciferase (mt-luc) RNA will be an excellent reporter for revealing cis-acting elements essential for in organello RNA processing, maturation and expression. Additionally, the mt-luc gene can be readily incorporated into any novel mitochondrial transducing vectors to assess intra-organellar transcription and translation.

An mtDNA mutation in the initiation codon of the cytochrome C oxidase subunit II gene results in lower levels of the protein and a mitochondrial encephalomyopathy.

A novel heteroplasmic 7587T-->C mutation in the mitochondrial genome which changes the initiation codon of the gene encoding cytochrome c oxidase subunit II (COX II), was found in a family with mitochondrial disease. This T-->C transition is predicted to change the initiating methionine to threonine. The mutation load was present at 67% in muscle from the index case and at 91% in muscle from the patient's clinically affected son. Muscle biopsy samples revealed isolated COX deficiency and mitochondrial proliferation. Single-muscle-fiber analysis revealed that the 7587C copy was at much higher load in COX-negative fibers than in COX-positive fibers. After microphotometric enzyme analysis, the mutation was shown to cause a decrease in COX activity when the mutant load was >55%-65%. In fibroblasts from one family member, which contained >95% mutated mtDNA, there was no detectable synthesis or any steady-state level of COX II. This new mutation constitutes a new mechanism by which mtDNA mutations can cause disease-defective initiation of translation.

MHC class I expression protects target cells from lysis by Ly49-deficient fetal NK cells.

Using appropriate conditions natural killer (NK) cells can be cultured from the liver and thymus of day 14 fetal mice. These fetal NK cells are phenotypically and functionally indistinguishable from adult NK cells with the exception that they lack measurable expression of all of the Ly49 molecules that can currently be detected with antibodies. Despite this, they preferentially kill tumor cells and blast cells deficient in the expression of major histocompatibility complex class I molecules, although the degree of discrimination is usually weaker than that shown by adult NK cells and varies depending on the particular combination of effector and target cells used. Polymerase chain reaction analysis revealed that although fetal NK cells are severely deficient in the expression of mRNA for Ly49A, B, C, D, G, H, and I they express high levels of Ly49E mRNA, raising the possibility that Ly49E may have an important and special function in the early development of the NK lineage.

Intracellular mitochondrial triplasmy in a patient with two heteroplasmic base changes.

We report the clinical, biochemical, and genetic investigation of a patient with a severe mitochondrial encephalomyopathy. Genetic studies identified a novel, heteroplasmic tRNA mutation at nt 10010. This T-->C transition is located in the DHU loop of mitochondrial tRNA(Gly). In skeletal muscle, it was present at lower levels in cytochrome c oxidase (COX)-normal (87.2% +/- 11%) compared with COX-deficient fibers (97.3% +/- 2.6%); it was found in skin fibroblasts and blood cells, but at lower levels of heteroplasmy (15% +/- 6% and 17% +/- 10%, respectively). A second, heteroplasmic transition (A-->G), at nt 5656, showed a different distribution than the tRNA(Gly) mutation, with very low levels in skeletal muscle (< 3%) but higher levels in blood (22.7% +/- 3%) and skin fibroblasts (21% +/- 2%). These transitions were followed both in vivo, by repeat biopsy and blood sampling, and in vitro, by establishing primary cultures of myoblasts and skin fibroblasts. Repeat muscle biopsy showed a dramatic increase in COX-deficient fibers, but not of the tRNAGly mutation. Indeed, no significant change in heteroplasmy was measured for either substitution in muscle or blood. In vitro analysis gave very different results. The T10010C was not found in cultured myoblasts, even at early passage. In uncloned fibroblasts, the T10010C was stable (approximately 10%) for several passages but then gradually was lost. In contrast, the A5656G rose progressively from 27% to 91%. In cloned fibroblasts, different combinations of both base-pair changes and wild type could be identified, confirming the presence of clonal, intracellular triplasmy.

The mRNA-binding protein COLBP is glutamate dehydrogenase.

Expression of the liver-type isopeptides of cytochrome c oxidase is regulated post-transcriptionally. An RNA-binding activity has been found in only those cells where the liver-type subunits are detected. This binding protein, termed COLBP, recognises sequences or structures within the 3'-untranslated regions of transcripts encoding these liver-type isopeptides and has been implicated in the modulation of mRNA expression. We now show by subcellular fractionation, immunocompetition, UV-crosslinking and shift-Western studies that the metabolic enzyme glutamate dehydrogenase, previously reported as being able to bind RNA, is the cytochrome c oxidase transcript-binding protein, COLBP.

Gene therapy for mitochondrial DNA defects: is it possible?

Defects of the mitochondrial genome are increasingly being recognised as important causes of disease. Patients may present at any age and the symptoms vary from fatal lactic acidosis in infancy to muscle disease in adults. For most patients there is no satisfactory treatment and there is a gradual deterioration leading to severe disability and death. In the absence of any biochemical treatment, gene therapy must be considered for these patients. This review addresses the unique problems associated with the treatment of defects of the mitochondrial genome by gene therapy, and discusses the approaches which we believe may be of value.

Inhibition of mitochondrial protein synthesis promotes increased stability of nuclear-encoded respiratory gene transcripts.

To investigate the molecular basis of nuclear-mitochondrial communication, we have been studying the effect of mitochondrial stress (stimulated by inhibition of mitochondrial protein synthesis) on the homeostasis of transcripts encoding nuclear and mitochondrial gene products. We report that in cells treated with the inhibitor thiamphenicol, nuclear-encoded respiratory gene transcripts were dramatically stabilized. A concomitant up-regulation in the activity of the only known respiratory transcript binding protein, cytochrome c oxidase L-form transcript binding protein (COLBP), was also noted in thiamphenicol-treated cells, demonstrating a potential mechanism for the increased transcript protection. In contradistinction, stability of all mitochondrial RNAs was unaffected by the inhibitor, as were the nuclear-encoded beta-actin, alpha-tubulin mRNAs and total cytosolic RNA. Steady state levels of all nuclear-encoded transcripts tested remained constant after inhibition of mitochondrial protein synthesis, whereas a generalized increase in the levels of processed mitochondrial mRNA was noted. We conclude that thiamphenicol induces (i) an increase in steady state levels of mitochondrial mRNA, (ii) a selective protection of nuclear respiratory gene transcripts against degradation, and (iii) an up-regulation in activity of the respiratory transcript binding protein COLBP, consistent with this protein mediating increased transcript stability. Our results demonstrate a coordinated series of intracellular responses to thiamphenicol-induced mitochondrial stress, regulated at both the pre- and post-transcriptional levels.