Regulation Of Mitochondrial Protein Synthesis Research Articles

Aspartyl-tRNA synthetase 2 orchestrates iron-sulfur metabolism in hematopoietic stem cells via fine-tuning alternative RNA splicing

Aspartyl-tRNA synthetase 2 (Dars2) is involved in the regulation of mitochondrial protein synthesis and tissue-specific mitochondrial unfolded protein response (UPRmt). The role of Dars2 in the self-renewal and differentiation of hematopoietic stem cells (HSCs) is unknown. Here, we show that knockout (KO) of Dars2 significantly impairs the maintenance of hematopoietic stem and progenitor cells (HSPCs) without involving its tRNA synthetase activity. Dars2 KO results in significantly reduced expression of Srsf2/3/6 and impairs multiple events of mRNA alternative splicing (AS). Dars2 directly localizes to Srsf3-labeled spliceosomes in HSPCs and regulates the stability of Srsf3. Dars2-deficient HSPCs exhibit aberrant AS of mTOR and Slc22a17. Dars2 KO greatly suppresses the levels of labile ferrous iron and iron-sulfur cluster-containing proteins, which dampens mitochondrial metabolic activity and DNA damage repair pathways in HSPCs. Our study reveals that Dars2 plays a crucial role in the iron-sulfur metabolism and maintenance of HSPCs by modulating RNA splicing.

Phosphorylation of mammalian mitochondrial EF-Tu by Fyn and c-Src kinases

Src Family Kinases (SFKs) are tyrosine kinases known to regulate glucose and fatty acid metabolism as well as oxidative phosphorylation (OXPHOS) in mammalian mitochondria. We and others discovered the association of the SFK kinases Fyn and c-Src with mitochondrial translation components. This translational system is responsible for the synthesis of 13 mitochondrial (mt)-encoded subunits of the OXPHOS complexes and is, thus, essential for energy generation. Mitochondrial ribosomal proteins and various translation elongation factors including Tu (EF-Tumt) have been identified as possible Fyn and c-Src kinase targets. However, the phosphorylation of specific residues in EF-Tumt by these kinases and their roles in the regulation of protein synthesis are yet to be explored. In this study, we report the association of EF-Tumt with cSrc kinase and mapping of phosphorylated Tyr (pTyr) residues by these kinases. We determined that a specific Tyr residue in EF-Tumt at position 266 (EF-Tumt-Y266), located in a highly conserved c-Src consensus motif is one of the major phosphorylation sites. The potential role of EF-Tumt-Y266 phosphorylation in regulation of mitochondrial translation investigated by site-directed mutagenesis. Its phosphomimetic to Glu residue (EF-Tumt-E266) inhibited ternary complex (EF-Tumt•GTP•aatRNA) formation and translation in vitro. Our findings along with data mining analysis of the c-Src knock out (KO) mice proteome suggest that the SFKs have possible roles for regulation of mitochondrial protein synthesis and oxidative energy metabolism in animals.

Mitochondrial microRNAs: A Putative Role in Tissue Regeneration.

Simple SummaryDistinct tissue engineering strategies are currently being developed. Cell-based therapies, implantation of synthetic scaffolds or combination of a scaffold with seeded cells are being used according to the biologic premises. Mitochondria play a central role in cell life cycle due to their preponderant role in energy production. Recent data suggest that small non-coding RNAs encoded by mitochondria DNA such as microRNAs regulate a plethora of factors and consequently signaling pathways, crucial in disease pathogenesis and also putatively in regenerative processes. Unveiling mitochondrial microRNAs biological function and their targets will propel the development of innovative therapeutic and diagnostic tools.The most famous role of mitochondria is to generate ATP through oxidative phosphorylation, a metabolic pathway that involves a chain of four protein complexes (the electron transport chain, ETC) that generates a proton-motive force that in turn drives the ATP synthesis by the Complex V (ATP synthase). An impressive number of more than 1000 mitochondrial proteins have been discovered. Since mitochondrial proteins have a dual genetic origin, it is predicted that ~99% of these proteins are nuclear-encoded and are synthesized in the cytoplasmatic compartment, being further imported through mitochondrial membrane transporters. The lasting 1% of mitochondrial proteins are encoded by the mitochondrial genome and synthesized by the mitochondrial ribosome (mitoribosome). As a result, an appropriate regulation of mitochondrial protein synthesis is absolutely required to achieve and maintain normal mitochondrial function. Regarding miRNAs in mitochondria, it is well-recognized nowadays that several cellular mechanisms involving mitochondria are regulated by many genetic players that originate from either nuclear- or mitochondrial-encoded small noncoding RNAs (sncRNAs). Growing evidence collected from whole genome and transcriptome sequencing highlight the role of distinct members of this class, from short interfering RNAs (siRNAs) to miRNAs and long noncoding RNAs (lncRNAs). Some of the mechanisms that have been shown to be modulated are the expression of mitochondrial proteins itself, as well as the more complex coordination of mitochondrial structure and dynamics with its function. We devote particular attention to the role of mitochondrial miRNAs and to their role in the modulation of several molecular processes that could ultimately contribute to tissue regeneration accomplishment.

Uncovering the exercise‐related proteome signature in skeletal muscle

Exercise training has been recommended as a nonpharmacological strategy for the prevention and attenuation of skeletal muscle atrophy in distinct pathophysiological conditions. Despite the well-established phenotypic alterations, the molecular mechanisms underlying exercise-induced skeletal muscle remodeling are poorly characterized. Proteomics based on mass spectrometry have been successfully applied for the characterization of skeletal muscle proteome, representing a pivotal approach for the wide characterization of the molecular networks that lead to skeletal muscle remodeling. Nevertheless, few studies were performed to characterize the exercise-induced proteome remodeling of skeletal muscle, with only six research papers focused on the cross-talk between exercise and pathophysiological conditions. In order to add new insights on the impact of distinct exercise programs on skeletal muscle proteome, molecular network analysis was performed with bioinformatics tools. This analysis highlighted an exercise-related proteome signature characterized by the up-regulation of the capacity for ATP generation, oxygen delivery, antioxidant capacity and regulation of mitochondrial protein synthesis. Chronic endurance training up-regulates the tricarboxylic acid cycle and oxidative phosphorylation system, whereas the release of calcium ion into cytosol and amino acid metabolism are the biological processes up-regulated by a single bout of exercise. Other issues as exercise intensity, load, mode and regimen as well as muscle type also influence the exercise-induced proteome signature. The comprehensive analysis of the molecular networks modulated by exercise training in health and disease, taking in consideration all these variables, might not only support the therapeutic effect of exercise but also highlight novel targets for the development of enhanced pharmacological strategies.

Organization and Regulation of Mitochondrial Protein Synthesis.

Mitochondria are essential organelles of endosymbiotic origin that are responsible for oxidative phosphorylation within eukaryotic cells. Independent evolution between species has generated mitochondrial genomes that are extremely diverse, with the composition of the vestigial genome determining their translational requirements. Typically, translation within mitochondria is restricted to a few key subunits of the oxidative phosphorylation complexes that are synthesized by dedicated ribosomes (mitoribosomes). The dramatically rearranged mitochondrial genomes, the limited set of transcripts, and the need for the synthesized proteins to coassemble with nuclear-encoded subunits have had substantial consequences for the translation machinery. Recent high-resolution cryo-electron microscopy has revealed the effect of coevolution on the mitoribosome with the mitochondrial genome. In this review, we place the new structural information in the context of the molecular mechanisms of mitochondrial translation and focus on the novel ways protein synthesis is organized and regulated in mitochondria.

Mitochondriale Logistik: Assemblierung der Cytochrom-c-Oxidase

Mitochondrial respiratory-chain complexes assemble from subunits either translated in mitochondria or imported from the cytosol. In the early steps of respiratory chain biogenesis, specific assembly factors participate in the formation of assembly intermediates of the individual complexes. These intermediates play a critical role in feedback regulation of mitochondrial protein synthesis in response to the assembly state.

A Newly Identified Protein Regulates Translation in Mammalian Mitochondria

Mitochondria are responsible for providing 90% of the energy in eukaryotic organisms by oxidative phosphorylation (OXPHOS). Mitochondrially encoded components of OXPHOS complexes are all essential and synthesized by a specialized translation system containing 55S ribosomes in mitochondria. In the reevaluation of mitochondrial ribosomal proteins (MRPs) and ribosome‐interacting proteins, we identified Gadd45GIP1 as a bona fide component in purified 55S ribosomes. Here, we report Gadd45GIP1 association with the large subunit of mammalian mitochondrial ribosomes and its role in the regulation of mitochondrial protein synthesis. In siRNA knock‐down studies, reduced expression of Gadd45GIP1 decreased de novo synthesis of the 13 mitochondrially encoded proteins of OXPHOS complexes in vivo. This indicates that the Gadd45GIP1 is an essential member of the mitochondrial ribosome, and its specific role in protein synthesis is currently under investigation.Supported by NIH grant R01GM071034 (E.C.K.)

NAD+‐dependent deacetylase SIRT3 regulates mitochondrial protein synthesis by deacetylation of mitochondrial ribosomal protein L10

Reversible acetylation regulates many biological processes including mitochondrial energy metabolism, cell survival and longevity. A member of the sirtuin family of NAD+‐dependent deacetylases, SIRT3, is important for regulation of reversible acetylation in mammalian mitochondria. In this study, we identified ribosomal MRPL10 as one of the major acetylated proteins in the mitochondrial ribosome. Ribosome associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD+‐dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site‐directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 lead to an increase in translational activity of mitochondrial ribosomes in SIRT3−/− mice. In a similar manner, ectopic expression and knock‐down of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD+‐ dependent deacetylase, SIRT3.Supported by NIH R01s to ECK (GM071034), YB (AG025223), and QT (DK075978).

NAD+-dependent Deacetylase SIRT3 Regulates Mitochondrial Protein Synthesis by Deacetylation of the Ribosomal Protein MRPL10

A member of the sirtuin family of NAD(+)-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated protein in the mitochondrial ribosome. Ribosome-associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD(+)-dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site-directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 led to an increase in translational activity of mitochondrial ribosomes in Sirt3(-/-) mice. In a similar manner, ectopic expression and knockdown of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD(+)-dependent deacetylase, SIRT3.

Nuclear respiratory factor 2 activates transcription of human mitochondrial translation initiation factor 2 gene

We studied the transcriptional regulation of the human mitochondrial translation initiation factor 2 (IF2mt) gene. The minimal promoter region for the human IF2mt gene contains binding sites for Nuclear Respiratory Factor 2 (NRF-2), which is often involved in the transcription of mitochondrial-related genes. Electrophoresis mobility shift assay (EMSA) analyses indicated that NRF-2α/β binds to the IF2mt promoter. Reporter assays, where HEK293T cells were co-transfected with an NRF-2α/β-expressing vector and/or an IF2mt promoter reporter vector, revealed that NRF-2 trans-activates the IF2mt promoter. NRF-2 sites were also found in the promoters of several other mitochondrial translation factors, which suggests NRF-2 may play a key role in the regulation of mitochondrial protein synthesis.

Phenotypic expression of mtDNA heteroplasmy in the skeletal muscle of patients with oculomyopathy: Defect in mitochondrial protein synthesis

The biochemical consequences of mtDNA heteroplasmy, observed in patients with a range of diseases associated with the mitochondrial respiratory enzymes deficiency is of particular interest, as they might provide information with regard to the regulatory interactions which govern the expression of the human mitochondrial genome. Three patients with chronic progressive external ophthalmoplegia (CPEO) were investigated to study the consequences of mtDNA heteroplasmy on mitochondrial protein synthesis. All 3 patients exhibited partially deleted mtDNA species (varying in size from 10.5 to 14 kb) in their skeletal muscle, which co-existed with the normal 16.5 kb mtDNA. The examination of mitochondrial translation products following the incorporation of [ 35S]methionine by isolated mitochondria, showed grossly abnormal patterns of mitochondrial translation products, suggesting a major disturbance in the regulation of mitochondrial protein synthesis.

Isolation and incubation conditions to study heart mitochondrial protein synthesis

Although much is now known with regard to the processes of mammalian mitochondrial gene expression, relatively little is known concerning the quantitative regulation of this pathway in response to hormones or other physiological stimuli. This has been caused, in large part, by the lack of adequate assay systems in which such processes can be meaningfully measured. The purpose of this and the companion paper [E. E. McKee, B. L. Grier, G. S. Thompson, A. C. F. Leung, and J. D. McCourt. Am. J. Physiol. 258 [Endocrinol. Metab. 21):E503-E510, 1990] is to describe a system in which the quantitative regulation of mitochondrial protein synthesis in rat heart can be investigated. In this report the conditions for mitochondrial isolation and labeling are described, and the importance of isolating intact, tightly coupled mitochondria in obtaining high and reliable rates of protein synthesis is demonstrated. The highest levels of protein synthesis are obtained in mitochondria isolated from hearts perfused and homogenized in the presence of subtilisin, conditions in which the fastest rates of state 3 respiration and the highest respiratory control ratios are also observed. Analysis of the free amino acid pools indicates that isolated heart mitochondria have a negligible level of endogenous methionine as well as other amino acids. As a result, the concentration and specific radioactivity of the [35S]methionine pool serving protein synthesis could be easily determined. Optimal translation occurred at 30 degrees C at a pH of 7.0-7.2 and required the addition of methionine (20 microM), the other 19 amino acids (0.1 mM each), K+ (60-90 mM), Cl- (30-90 mM), Mg2+ (0.5-5 mM), and bovine serum albumin (1 mg/ml). As shown in the companion paper, adenine nucleotide (0.5-4.0 mM) and oxidizable substrate (10-20 mM glutamate) are also required for isolated heart mitochondrial protein synthesis. Analysis of labeled mitochondrial translation products demonstrated that bona fide mitochondrial peptides were synthesized.

Regulation of biosynthesis of the rat liver inner mitochondrial membrane by thyroid hormone

Regulation of mitochondrial protein synthesis by thyroid hormone has been studied in isolated rat hepatocytes and liver mitochondria. Small doses (5 μg/100 g body wt) of triiodothyronine (T 3) injected into hypothyroid rats increased both state 3 and 4 respiration by approximately 100%, while the ADP:O ratio remained constant. This suggests that T 3 increases the numbers of functional respiratory chain units. T 3 also induces mitochondrial protein synthesis by 50–100%. Analysis of the mitochondrial translation products show that all of the products were induced. No differential translation of the peptides involved in the respiratory chain was found. Regulation of the cytoplasmically made inner membrane peptides was also investigated in isolated hepatocytes. The majority of these peptides were not influenced by T 3, in contrast to the finding with mitochondrial translation products. Those found to be regulated by T 3 belong to two subsets, which were either induced or repressed by hormone. Thus, T 3 timulated a general increase in the synthesis of mitochondrially translated inner membrane peptides, but regulates selectively those inner membrane peptides translated on cytoplasmic ribosomes. The findings suggest that hormone regulation of the respiratory chain is exerted through a few selective proteins, perhaps those which require subunits made from both nuclear and mitochondrial genes.

Regulation of mitochondrial protein synthesis by cytoplasmic proteins.

Isolated yeast mitochondria, which synthesize identifiable polypeptides identical to those made in vivo, have been used in an invitro system to study cytoplasmic control of mitochondrial protein synthesis. It has been found that protein synthesis in isolated mitochondria is dependent on an endogenous pool of cytoplasmically synthesized proteins present within mitochondria at the time of isolation, that protein synthesis ceases apparently when this pool of proteins is depleted, and that a cytoplasmic extract can restore protein synthesis in depleted mitochondria. By use of depleted mitochondria to assay for stimulatory factors it has been found that the bulk of the stimulatory activity in the cytoplasm is of a protein nature and resides predominantly in the postpolysomal supernatant. At least one cytoplasmic stimulatory protein appears to exert a specific effect on the synthesis of subunits I-III of cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase; EC 1.9.3.1).

Regulation of mitochondrial protein synthesis at the polyribosomal level.

Polysomes consisting of two to eight monosomes were isolated from yeast mitochondria by lysing the mitochondria with Triton X-100 and centrifugation in a 20 to 40% linear sucrose gradient. When yeast spheroplasts were pulse-labeled with [3H]-Leucine in the presence of cycloheximide to block cytoplasmic protein synthesis, radioactivity which was trichloroacetic acid-precipitable was present mainly in the polysome region. Incorporation of leucine was blocked by erythromycin, a specific inhibitor of mitochondrial protein synthesis. Release of radioactivity to the top of the gradient resulted from treating labeled polysomes with either puromycin or ribonuclease (in the latter case with the breakdown of polysomes), indicating that the radioactivity was present in nascent polypeptide chains. Yeast cells were grown in chloramphenicol for 3 hours and in fresh medium for 1 hour and then pulse-labeled with either [3H]leucine or [14C]formate. Three parameters showed a 2-fold increase in cells grown in chloramphenicol prior to pulse labeling: the polysome to monosome ratio, the amount of labeled precursor incorporated into proteins, and the rate of polypeptide chain initiation as judged by the formation of fMet-puromycin. Conversely, these parameters were all decreased approximately 50% in cells treated with cycloheximide prior to pulse labeling. Mitochondria were also isolated from cells previously grown in chloramphenicol or cycloheximide and incubated in vitro with [3H]leucine under optimal conditions. Acid-precipitable radioactivity in the polysome region was increased 3-fold in mitochondria from cells grown previously in chloramphenicol and decreased 75% in those grown in cycloheximide. Furthermore, chain initiation was deomonstrated in the isolated mitochondria by formation of fMet-puromycin. The rate of chain initiation in vitro was increased 2-fold in mitochondria isolated from chloramphenicol-treated cells.

Stimulation of mitochondrial protein synthesis by metal ions

1–10 μM Cu 2+, Ag +, and Au 3+ were found to stimulate rat liver mitochondrial protein synthesis in vitro. Cu 2+ and Ag + also produced an increase in mitochondrial volume (“swelling”). Thus, thyroid hormones and their analogs are not unique, as suggested previously (Buchanan, J.L., Primack, M.P. and Tapley, D.F. (1970) Endocrinology 87, 993–999), in stimulating both mitochondrial protein synthesis and swelling. Furthermore, the data suggest a role for Cu 2+ in the regulation of mitochondrial protein synthesis.