Translation Of Mitochondrial mRNAs Research Articles

LRPPRC is necessary for polyadenylation and coordination of translation of mitochondrial mRNAs

Regulation of mtDNA expression is critical for maintaining cellular energy homeostasis and may, in principle, occur at many different levels. The leucine-rich pentatricopeptide repeat containing (LRPPRC) protein regulates mitochondrial mRNA stability and an amino-acid substitution of this protein causes the French-Canadian type of Leigh syndrome (LSFC), a neurodegenerative disorder characterized by complex IV deficiency. We have generated conditional Lrpprc knockout mice and show here that the gene is essential for embryonic development. Tissue-specific disruption of Lrpprc in heart causes mitochondrial cardiomyopathy with drastic reduction in steady-state levels of most mitochondrial mRNAs. LRPPRC forms an RNA-dependent protein complex that is necessary for maintaining a pool of non-translated mRNAs in mammalian mitochondria. Loss of LRPPRC does not only decrease mRNA stability, but also leads to loss of mRNA polyadenylation and the appearance of aberrant mitochondrial translation. The translation pattern without the presence of LRPPRC is misregulated with excessive translation of some transcripts and no translation of others. Our findings point to the existence of an elaborate machinery that regulates mammalian mtDNA expression at the post-transcriptional level.

The effect of mutated mitochondrial ribosomal proteins S16 and S22 on the assembly of the small and large ribosomal subunits in human mitochondria

Mutations in mitochondrial small subunit ribosomal proteins MRPS16 or MRPS22 cause severe, fatal respiratory chain dysfunction due to impaired translation of mitochondrial mRNAs. The loss of either MRPS16 or MRPS22 was accompanied by the loss of most of another small subunit protein MRPS11. However, MRPS2 was reduced only about 2-fold in patient fibroblasts. This observation suggests that the small ribosomal subunit is only partially able to assemble in these patients. Two large subunit ribosomal proteins, MRPL13 and MRPL15, were present in substantial amounts suggesting that the large ribosomal subunit is still present despite a non-functional small subunit.

A Single Mammalian Mitochondrial Translation Initiation Factor Functionally Replaces Two Bacterial Factors

The mechanism of translation in eubacteria and organelles is thought to be similar. In eubacteria, the three initiation factors IF1, IF2, and IF3 are vital. Although the homologs of IF2 and IF3 are found in mammalian mitochondria, an IF1 homolog has never been detected. Here, we show that bovine mitochondrial IF2 (IF2(mt)) complements E. coli containing a deletion of the IF2 gene (E. coli DeltainfB). We find that IF1 is no longer essential in an IF2(mt)-supported E. coli DeltainfB strain. Furthermore, biochemical and molecular modeling data show that a conserved insertion of 37 amino acids in the IF2(mt) substitutes for the function of IF1. Deletion of this insertion from IF2(mt) supports E. coli for the essential function of IF2. However, in this background, IF1 remains essential. These observations provide strong evidence that a single factor (IF2(mt)) in mammalian mitochondria performs the functions of two eubacterial factors, IF1 and IF2.

Overexpression of the COX2 translational activator, Pet111p, prevents translation of COX1 mRNA and cytochrome c oxidase assembly in mitochondria of Saccharomyces cerevisiae

Dramatically elevated levels of the COX2 mitochondrial mRNA-specific translational activator protein Pet111p interfere with respiratory growth and cytochrome c oxidase accumulation. The respiratory phenotype appears to be caused primarily by inhibition of the COX1 mitochondrial mRNA translation, a finding confirmed by lack of cox1Delta::ARG8(m) reporter mRNA translation. Interference with Cox1p synthesis depends to a limited extent upon increased translation of the COX2 mRNA, but is largely independent of it. Respiratory growth is partially restored by a chimeric COX1 mRNA bearing the untranslated regions of the COX2 mRNA, and by overproduction of the COX1 mRNA-specific activators, Pet309p and Mss51p. These results suggest that excess Pet111p interacts unproductively with factors required for normal COX1 mRNA translation. Certain missense mutations in PET111 alleviate the interference with COX1 mRNA translation but do not completely restore normal respiratory growth in strains overproducing Pet111p, suggesting that elevated Pet111p also perturbs assembly of newly synthesized subunits into active cytochrome c oxidase. Thus, this severe imbalance in translational activator levels appears to cause multiple problems in mitochondrial gene expression, reflecting the dual role of balanced translational activators in cooperatively regulating both the levels and locations of organellar translation.

Reconstitution of uridine-deletion precleaved RNA editing with two recombinant enzymes

Uridine insertion/deletion RNA editing in trypanosomatid mitochondria is a posttranscriptional RNA modification phenomenon required for translation of mitochondrial mRNAs. This process involves guide RNA-mediated cleavage at a specific site, insertion or deletion of Us from the 3' end of the 5' mRNA fragment, and ligation of the two mRNA fragments. The Leishmania major RNA ligase-containing complex protein 2 expressed in insect cells has a 3'-5' exoribonuclease activity and was therefore renamed RNA editing exonuclease 1 (REX1). Recombinant REX1 specifically trims 3' overhanging Us and stops at a duplex region. Evidence is presented that REX1 is responsible for deletion of the 3' overhanging Us from the bridged mRNA 5' cleavage fragment and that RNA editing ligase 1 is responsible for the ligation of the two mRNA cleavage fragments in U-deletion editing. The evidence involves both in vivo down-regulation of REX1 expression in Trypanosoma brucei by RNA interference and the reconstitution of precleaved U-deletion in vitro editing with only two recombinant enzymes: recombinant REX1 and recombinant RNA editing ligase 1.

"Ping-pong" interactions between mitochondrial tRNA import receptors within a multiprotein complex.

The mitochondrial genomes of a wide variety of species contain an insufficient number of functional tRNA genes, and translation of mitochondrial mRNAs is sustained by import of nucleus-encoded tRNAs. In Leishmania, transfer of tRNAs across the inner membrane can be regulated by positive and negative interactions between them. To define the factors involved in such interactions, a large multisubunit complex (molecular mass, approximately 640 kDa) from the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania, consisting of approximately 130-A particles, was isolated. The complex, when incorporated into phospholipid vesicles, induced specific, ATP- and proton motive force-dependent transfer of Leishmania tRNA(Tyr) as well as of oligoribonucleotides containing the import signal YGGYAGAGC. Moreover, allosteric interactions between tRNA(Tyr) and tRNA(Ile) were observed in the RNA import complex-reconstituted system, indicating the presence of primary and secondary tRNA binding sites within the complex. By a combination of antibody inhibition, photochemical cross-linking, and immunoprecipitation, it was shown that binding of tRNA(Ile) to a 21-kDa component of the complex is dependent upon tRNA(Tyr), while binding of tRNA(Tyr) to a 45-kDa component is inhibited by tRNA(Ile). This "ping-pong" mechanism may be an effective means to maintain a balanced tRNA pool for mitochondrial translation.

Increased synthesis and decreased stability of mitochondrial translation products in yeast as a result of loss of mitochondrial (NAD +)-dependent isocitrate dehydrogenase

We have previously demonstrated that the yeast Krebs cycle enzyme NAD +-dependent isocitrate dehydrogenase (Idh) binds specifically and with high affinity to the 5′-untranslated leader sequences of mitochondrial mRNAs in vitro and have proposed a role for the enzyme in the regulation of mitochondrial translation [Elzinga, S.D.J. et al. (2000) Curr. Genet., in press]. Although our studies initially failed to reveal any consistent correlation between idh disruption and mitochondrial translational activity, it is now apparent that compensatory extragenic suppressor mutations readily accumulate in idh disruption strains thereby masking mutant behaviour. Now, pulse-chase protein labelling of isolated mitochondria from an Idh disruption mutant lacking suppressor mutations reveals a strong (2–3-fold) increase in the synthesis of mitochondrial translation products. Strikingly, the newly synthesised proteins are more short-lived than in mitochondria from wild-type cells, their degradation occurring with a 2–3-fold reduced half-life. Enhanced degradation of translation products is also a feature of yeast mutants in which tethering/docking of mitochondrial mRNAs is disturbed. We therefore suggest that binding of Idh to mitochondrial mRNAs may suppress inappropriate translation of mitochondrial mRNAs.

3 Chaperoning Mitochondrial Biogenesis

I. INTRODUCTION The parallel development of powerful in vitro systems and of genetic approaches has allowed considerable progress in understanding the mechanisms of protein transport into various cellular compartments. It is becoming ever more evident that transport processes across different cellular membranes are based on similar principles. Polypeptide chains appear to traverse lipid bilayers through proteinaceous pores. Translocation requires a “translocation-competent,” rather unfolded conformation. As a consequence, proteins must be partially unfolded or kept in an unfolded conformation prior to the translocation event and must refold after crossing the lipid membrane bilayer. In recent years, increasing evidence was obtained that both represent assisted processes. Molecular chaperones, in many cases originally identified as heat shock proteins, modulate the folding state of polypeptide chains in different cellular compartments. Mitochondria, which contain heat shock proteins of the hsp70 and hsp60 family, proved to be a useful model system to study the function of chaperone proteins in protein translocation and folding. Although mitochondria contain their own DNA and independent systems for replication and protein synthesis, only a few subunits of the oxidative phosphorylation system and, in some organisms, components mediating splicing and translation of mitochondrial mRNA are encoded by the mitochondrial genome (Grivell 1989). About 95% of the total mass of mitochondrial proteins are encoded in the nucleus. They are synthesized on cytosolic polyribosomes, many of them as precursor molecules with amino-terminal presequences containing the targeting information. Import can occur posttranslationally followed by sorting to the various sub-compartments of mitochondria, the outer and inner...

In-vitro translation of mitochondrial mRNAs by yeast mitochondrial ribosomes is hampered by the lack of start-codon recognition.

In an attempt to reconstitute an homologous in-vitro translation system for yeast mitochondrial mRNAs, we have isolated ribosomes, supernatant factors, and tRNAs from mitochondria of Saccharomyces carlsbergensis. While poly(U) is translated faithfully in this system, no translation of in-vitro synthesised cytochrome c oxidase subunit II (COX2) mRNA could be detected. Formation of formylmethionyl-puromycin on mitochondrial ribosomes is stimulated by ApUpG, but not by COX2 mRNA, although mitochondrial small ribosomal subunits bind to this mRNA in vitro, even without added tRNA and initiation factors. We conclude, therefore, that the inability to faithfully translate mitochondrial mRNAs in vitro may be the result of an inability of mitochondrial ribosomes to recognize the initiation codon.

Genetic approaches to the study of mitochondrial biogenesis in yeast.

In contrast to most other organisms, the yeast Saccharomyces cerevisiae can survive without functional mitochondria. This ability has been exploited in genetic approaches to the study of mitochondrial biogenesis. In the last two decades, mitochondrial genetics have made major contributions to the identification of genes on the mitochondrial genome, the mapping of these genes and the establishment of structure-function relationships in the products they encode. In parallel, more than 200 complementation groups, corresponding to as many nuclear genes necessary for mitochondrial function or biogenesis have been described. Many of the latter are required for post-transcriptional events in mitochondrial gene expression, including the processing of mitochondrial pre-RNAs, the translation of mitochondrial mRNAs, or the assembly of mitochondrial translation products into the membrane. The aim of this review is to describe the genetic approaches used to unravel the intricacies of mitochondrial biogenesis and to summarize recent insights gained from their application.

The yeast nuclear gene CBS1 is required for translation of mitochondrial mRNAs bearing the cob 5' untranslated leader.

Mitochondrial translation of the cob mRNA to yield apocytochrome b is specifically dependent on the nuclear gene CBS1, while mitochondrial translation of the oxi2 mRNA to yield cytochrome oxidase subunit III (cox III) is specifically dependent on the nuclear gene PET494. Chimeric oxi2 mRNAs bearing the 5' leaders of other mitochondrial mRNAs, transcribed from rho- mitochondrial DNAs termed MSU494, are translated in pet494 mutants. In this study, we examined translation of coxIII from MSU494-encoded chimeric mRNAs in zygotes of defined nuclear and mitochondrial genotype. CoxIII was translated from a chimeric mRNA bearing the cob leader only when the zygotes contained a wild-type CBS1 gene. CoxIII translation from an mRNA bearing the 5' leader of the mitochondrial gene aap1 was not dependent on CBS1 activity. We conclude that the product of the nuclear gene CBS1, or something under its control, acts in the mitochondrion on the cob mRNA 5' leader to activate translation of down-stream coding sequences.

Endogenous read-through of a UGA termination codon in a Saccharomyces cerevisiae cell-free system: evidence for involvement of both a mitochondrial and a nuclear tRNA.

Globin mRNA, translated in a Saccharomyces cerevisiae cell-free protein synthesizing system prepared from a [psi+ rho+] strain, primarily directed the synthesis of alpha- and beta-globin. A third globin mRNA-specific polypeptide was also synthesized, representing approximately 10% of the total translation products. This polypeptide (beta') was synthesized by translational read-through of the beta- globin mRNA UGA terminator and was mediated primarily by an endogenous tRNA coded for by the mitochondria. This mitochondrial tRNA, when charged, could be preferentially bound, in high salt, to benzoylated DEAE-cellulose, a characteristic of a tRNATrp. The synthesis of beta- mediated by this mitochondrial tRNATrp was significantly reduced when the translation system was prepared from an isogenic [psi-] strain. Evidence for a nuclear-coded tRNA, also able to suppress the beta-globin mRNA UGA terminator in [psi+] but not [psi-] lysates, was also obtained. The presence of these endogenous UGA suppressor activities in the yeast cell-free system should allow successful in vitro translation of mitochondrial mRNAs.

Endogenous read-through of a UGA termination codon in a Saccharomyces cerevisiae cell-free system: evidence for involvement of both a mitochondrial and a nuclear tRNA.

Globin mRNA, translated in a Saccharomyces cerevisiae cell-free protein synthesizing system prepared from a [psi+ rho+] strain, primarily directed the synthesis of alpha- and beta-globin. A third globin mRNA-specific polypeptide was also synthesized, representing approximately 10% of the total translation products. This polypeptide (beta') was synthesized by translational read-through of the beta- globin mRNA UGA terminator and was mediated primarily by an endogenous tRNA coded for by the mitochondria. This mitochondrial tRNA, when charged, could be preferentially bound, in high salt, to benzoylated DEAE-cellulose, a characteristic of a tRNATrp. The synthesis of beta- mediated by this mitochondrial tRNATrp was significantly reduced when the translation system was prepared from an isogenic [psi-] strain. Evidence for a nuclear-coded tRNA, also able to suppress the beta-globin mRNA UGA terminator in [psi+] but not [psi-] lysates, was also obtained. The presence of these endogenous UGA suppressor activities in the yeast cell-free system should allow successful in vitro translation of mitochondrial mRNAs.

In vitro reconstruction of the mitochondrial translation system of yeast.

We have isolated the translation system from yeast mitochondria and have reconstructed it in vitro. This submitochondrial system, composed of mitochondrial ribosomes, tRNA, pH 5 fraction and mRNA, is maximally active at 10 mM Mg2+ and 100 mM KCl or NH4Cl. NH4+ is more stimulatory than K+. Added Escherichia coli tRNA gives less than half the activity obtained with added mitochondrial tRNA. Activity is enhanced with protease inhibitors but not with Ca2+, spermine, or spermidine. In contrast to heterologous translation systems, the present system produces products with molecular weights similar to those of products synthesized by yeast mitochondria in vivo and by intact yeast mitochondria in vitro. The results support the idea that the unique coding features of the mitochondrial genome require a unique translation system for accurate translation of mitochondrial mRNAs.

In vitro suppression of UGA codons in a mitochondrial mRNA.

Although both prokaryotic and eukaryotic messenger RNAs can be easily translated in heterologous protein-synthesizing systems, attempts to achieve correct synthesis of mitochondrial proteins by translation of mitochondrial mRNAs in such systems have failed. In general, the products of synthesis are of low molecular weight and presumably represent fragments of mitochondrial proteins. These fragments display a strong tendency to aggregate. Explanations have included the use by mitochondria of codons requiring a specialized tRNA population and the fortuitous occurrence within genes of purine-rich sequences resembling bacterial ribosome binding sites. In addition, the long 5'-leader sequences present in many mitochondrial (mt) RNAs may also contribute to difficulties in mRNA recognition by heterologous ribosomes. Recent sequence analysis of human mtDNA suggests that the genetic code used by mammalian mitochondria deviates in a number of respects from the 'universal' code, the most striking of these being the use of the UGA termination codon to specify tryptophan. That this may also apply in yeast mitochondria has been shown by Fox and Macino et al., thus providing an obvious and easily testable explanation for the inability of heterologous systems to synthesize full-length mitochondrial proteins. We confirm this explanation and describe here the in vitro synthesis of a full-length subunit II of yeast cytochrome c oxidase in a wheat-germ extract supplemented with a partially purified mitochondrial mRNA for this protein and a UGA-suppressor tRNA from Schizosaccharomyces pombe.