Regulation Of Mitochondrial Translation Research Articles

Fancd2-deficient hematopoietic stem and progenitor cells depend on augmented mitochondrial translation for survival and proliferation.

Members of the Fanconi anemia (FA) protein family are involved in multiple cellular processes including response to DNA damage and oxidative stress. Here we show that a major FA protein, Fancd2, plays a role in mitochondrial biosynthesis through regulation of mitochondrial translation. Fancd2 interacts with Atad3 and Tufm, which are among the most frequently identified components of the mitochondrial nucleoid complex essential for mitochondrion biosynthesis. Deletion of Fancd2 in mouse hematopoietic stem and progenitor cells (HSPCs) leads to increase in mitochondrial number, and enzyme activity of mitochondrion-encoded respiratory complexes. Fancd2 deficiency increases mitochondrial protein synthesis and induces mitonuclear protein imbalance. Furthermore, Fancd2-deficient HSPCs show increased mitochondrial respiration and mitochondrial reactive oxygen species. By using a cell-free assay with mitochondria isolated from WT and Fancd2-KO HSPCs, we demonstrate that the increased mitochondrial protein synthesis observed in Fancd2-KO HSPCs was directly linked to augmented mitochondrial translation. Finally, Fancd2-deficient HSPCs are selectively sensitive to mitochondrial translation inhibition and depend on augmented mitochondrial translation for survival and proliferation. Collectively, these results suggest that Fancd2 restricts mitochondrial activity through regulation of mitochondrial translation, and that augmented mitochondrial translation and mitochondrial respiration may contribute to HSC defect and bone marrow failure in FA.

A novel system to monitor mitochondrial translation in yeast.

The mitochondrial genome is responsible for the production of a handful of polypeptides that are core subunits of the membrane-bound oxidative phosphorylation system. Until now the mechanistic studies of mitochondrial protein synthesis inside cells have been conducted with inhibition of cytoplasmic protein synthesis to reduce the background of nuclear gene expression with the undesired consequence of major disturbances of cellular signaling cascades. Here we have generated a system that allows direct monitoring of mitochondrial translation in unperturbed cells. A recoded gene for superfolder GFP was inserted into the yeast (Saccharomyces cerevisiae) mitochondrial genome and enabled the detection of translation through fluorescence microscopy and flow cytometry in functional mitochondria. This novel tool allows the investigation of the function and regulation of mitochondrial translation during stress signaling, aging and mitochondrial biogenesis.

Plasticity of Mitochondrial Translation.

Mitochondria maintained a genome during evolution to synthesize core subunits of the oxidative phosphorylation system. Expression of the mitochondrial genome requires intraorganellar replication, transcription, and translation. Membrane-associated ribosomes translate mitochondrial-encoded proteins and facilitate co-translational insertion of newly synthesized polypeptides into the inner membrane. Considering that mitochondrial-encoded proteins assemble with imported, nuclear-encoded proteins into enzyme complexes of the oxidative phosphorylation system, it is expected that expression of mitochondrial genes should adapt to the availability of their nuclear-encoded partners. Recent work shows that mitochondrial translation is influenced by the cellular environment. We discuss how mitochondrial translation is affected by the cellular environment and propose models of translational plasticity that modulate mitochondrial translation in response to the availability of imported proteins.

Cardiomyocyte-specific loss of mitochondrial p32/C1qbp causes cardiomyopathy and activates stress responses.

Mitochondria are important organelles, dedicated to energy production. Mitochondrial p32/C1qbp, which functions as an RNA and protein chaperone, interacts with mitochondrial mRNA and is indispensable for mitochondrial function through its regulation of mitochondrial translation in cultured cell lines. However, the precise role of p32/C1qbp in vivo is poorly understood because of embryonic lethality in the systemic p32-deficient mouse. The goal of this study was to examine the physiological function of mitochondrial p32/C1qbp in the heart. We investigated the role of p32 in regulating cardiac function in mice using a Cre-loxP recombinase technology against p32 with tamoxifen-inducible knockdown or genetic ablation during postnatal periods. Cardiomyocyte-specific deletion of p32 resulted in contractile dysfunction, cardiac dilatation and cardiac fibrosis, compared with hearts of control mice. We also found decreased COX1 expression, decreased rates of oxygen consumption and increased oxidative stress, indicating that these mice had cardiac mitochondrial dysfunction provoked by p32-deficiency at early stage. Next, we investigated lifespan in cardiac-specific p32-deficient mice. The mice died beginning at 12 months and their median lifespan was ∼14 months. Cardiac mitochondria in the p32-deficient mice showed disordered alignment, enlargement and abnormalities in their internal structure by electron microscopy. We observed that, in p32-deficient compared with control myocytes, AMPKɑ was constitutively phosphorylated and 4EBP-1 and ribosomal S6K were less phosphorylated, suggesting impairment of mammalian target of rapamycin signalling. Finally, we found that expression levels of mitokines such as FGF21 and of integrated stress response genes were significantly increased. Metabolic analysis demonstrated that the urea cycle was impaired in the p32-deficient hearts. These findings support a key role for mitochondrial p32 protein in cardiac myocytes modulating mitochondrial translation and function, and thereby survival.

CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels.

Despite being one of the most studied proteases in bacteria, very little is known about the role of ClpXP in mitochondria. We now present evidence that mammalian CLPP has an essential role in determining the rate of mitochondrial protein synthesis by regulating the level of mitoribosome assembly. Through a proteomic approach and the use of a catalytically inactive CLPP, we produced the first comprehensive list of possible mammalian ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation, and a number of metabolic pathways. We further show that the defect in mitoribosomal assembly is a consequence of the accumulation of ERAL1, a putative 12S rRNA chaperone, and novel ClpXP substrate. The presented data suggest that the timely removal of ERAL1 from the small ribosomal subunit is essential for the efficient maturation of the mitoribosome and a normal rate of mitochondrial translation.

MTERF4 regulates the mitochondrial dysfunction induced by MPP+ in SH-SY5Y cells

Mitochondrial transcription termination factor 4, MTERF4, a member of the MTERF family, has been implicated in the regulation of mitochondrial translation by targeting NSUN4 to the large mitochondrial ribosome. Here, we found a novel role for MTERF4 in regulating mitochondrial dysfunction induced by MPP+. We observed that knockdown of MTERF4 in SH-SY5Y cells resulted in increased mitochondrial DNA transcription levels and decreased mitochondrial DNA translation levels. In addition, after treatment with 2 mM MPP+ for 24 h, the expression levels of MTERF4 were decreased compared to wide-type SH-SY5Y cells. Moreover, after exposure to 2 mM MPP+ for 24 h, knockdown of MTERF4 in SH-SY5Y cells worsened the mitochondrial dysfunction induced by MPP+, including increased reactive oxygen species, accumulated cleaved PARP-1, decreased mitochondrial membrane potential and depressed mitochondrial complexes. Furthermore, overexpression of MTERF4 in SH-SY5Y cells partially alleviated the mitochondrial dysfunction induced by MPP+. Based on these findings, we suggest that the main function of MTERF4 is regulating mtDNA expression, and it is the crucial factor in the mechanism of mitochondrial dysfunction in SH-SY5Y cells induced by MPP+. MTERF4 probably is the triggering of the pathogenesis of Parkinson's disease induced by environmental toxin.

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.

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.

The complete nucleotide sequence of theRattus norvegicus mitochondrial genome: Cryptic signals revealed by comparative analysis between vertebrates

This paper reports the nucleotide sequence of rat mitochondrial DNA, only the fourth mammalian mitochondrial genome to be completely sequenced. Extensive comparative studies performed with similar genomes from other organisms revealed a number of interesting features. 1) Messenger RNA genes: the codon strategy is mainly dictated by the base compositional constraints of the corresponding codogenic DNA strand. The usage of the initiation and termination codons follows well-established rules. In general the canonical initiator, ATG, and terminators, TAA and TAG (in rat, only TAA), are always present when there is gene overlapping or when the mRNAs possess untranslated nucleotides at the 5' or 3' ends. 2) Transfer RNA genes: a number of features suggest the peculiar evolutionary behavior of this class of genes and confirm their role in the duplication and rearrangement processes that took place in the evolution of the animal mitochondrial genome. 3) Ribosomal RNA genes: accurate sequence analysis revealed a number of significant examples of complementarity between ribosomal and messenger RNAs. This suggests that they might play an important role in the regulation of mitochondrial translation and transcription mechanisms. The properties revealed by our work shed new light on the organization and evolution of the vertebrate mitochondrial genome and more importantly open up the way to clearly aimed experimental studies of the regulatory mechanisms in mitochondria.

Regulation of mitochondrial membrane assembly in Neurospora crassa. Transient expression of a respiratory mutant phenotype.

Cultures of mutant cni-1, a chromosomal mutant of Neurospora crassa, undergo a marked change in respiratory properties as the age of the culture increases. Early log phase cultures have a high level of respiration that is insensitive to inhibition by cyanide or antimycin A. Late log and stationary phase cultures have reduced rates of respiration. A high percentage of this respiration is inhibited by cyanide. Mitochondria from early log phase cni-1 have an excess of cytochrome c and little or no detectable cytochrome aa3. Mitochondria from late log and stationary phase cultures have levels of c-, b-, and a-type cytochromes that are not significantly different in concentration from those found in wild type cells. The cytochrome aa3 content and the cytochrome oxidase activity of cni-1 mitochondria increase 5- to 10-fold as the age of the culture increases. Mitochondria from early log phase cells of cni-1 synthesize only polypeptides of apparent molecular weights 7,000 to 10,000 and donot synthesize any of the mitochondrial components of cytochrome oxidase. Mitochondria from late log and stationary phase cells synthesize the normal complement of mitochondrial translation products including the mitochondrial components of cytochrome oxidase. The assembly of cytochrome oxidase is likely due to the availability of the mitochondrially synthesized components of the enzyme. The regulation of mitochondrial translation in the cni-1 mutant is independent of the nutrient content of the growth medium and is due to the accumulation or depletion of some component within the cell.