[show abstract][hide abstract] ABSTRACT: Mitochondrial gene expression is predominantly regulated at the post-transcriptional level and mitochondrial ribonucleic acid (RNA)-binding proteins play a key role in RNA metabolism and protein synthesis. The AU-binding homolog of enoyl-coenzyme A (CoA) hydratase (AUH) is a bifunctional protein with RNA-binding activity and a role in leucine catabolism. AUH has a mitochondrial targeting sequence, however, its role in mitochondrial function has not been investigated. Here, we found that AUH localizes to the inner mitochondrial membrane and matrix where it associates with mitochondrial ribosomes and regulates protein synthesis. Decrease or overexpression of the AUH protein in cells causes defects in mitochondrial translation that lead to changes in mitochondrial morphology, decreased mitochondrial RNA stability, biogenesis and respiratory function. Because of its role in leucine metabolism, we investigated the importance of the catalytic activity of AUH and found that it affects the regulation of mitochondrial translation and biogenesis in response to leucine.
Nucleic Acids Research 03/2014; · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mammalian pentatricopeptide repeat domain (PPR) proteins are involved in regulation of mitochondrial RNA metabolism and translation and are required for mitochondrial function. We investigated an uncharacterised PPR protein, the supernumerary mitochondrial ribosomal protein of the small subunit 27 (MRPS27), and show that it associates with the 12S rRNA and tRNA(Glu), however it does not affect their abundance. We found that MRPS27 is not required for mitochondrial RNA processing or the stability of the small ribosomal subunit. However, MRPS27 is required for mitochondrial protein synthesis and its knockdown causes decreased abundance in respiratory complexes and cytochrome c oxidase activity. STRUCTURED SUMMARY OF PROTEIN INTERACTIONS: MRPS27 and MRPS15colocalize by cosedimentation through density gradient (View Interaction).
[show abstract][hide abstract] ABSTRACT: Human mitochondrial long noncoding RNAs (lncRNAs) have not been described to date. By analysis of deep-sequencing data we have identified three lncRNAs generated from the mitochondrial genome and confirmed their expression by Northern blotting and strand-specific qRT-PCR. We show that the abundance of these lncRNAs is comparable to their complementary mRNAs and that nuclear-encoded mitochondrial proteins involved in RNA processing regulate their expression. We also identify the 5' and 3' transcript ends of the three lncRNAs and show that mitochondrial RNase P protein 1 (MRPP1) is important for the processing of these transcripts. Finally, we show that mitochondrial lncRNAs form intermolecular duplexes and that their abundance is cell- and tissue-specific, suggesting a functional role in the regulation of mitochondrial gene expression.
[show abstract][hide abstract] ABSTRACT: Mammalian mitochondrial DNA is transcribed as precursor polycistronic transcripts containing 13 mRNAs, 2 rRNAs, punctuated by 22 tRNAs. The mechanisms involved in the excision of mitochondrial tRNAs from these polycistronic transcripts have remained largely unknown. We have investigated the roles of ELAC2, mitochondrial RNase P proteins 1 and 3, and pentatricopeptide repeat domain protein 1 in the processing of mitochondrial polycistronic transcripts. We used a deep sequencing approach to characterize the 5' and 3' ends of processed mitochondrial transcripts and provide a detailed map of mitochondrial tRNA processing sites affected by these proteins. We show that MRPP1 and MRPP3 process the 5' ends of tRNAs and the 5' unconventional, non tRNA containing site of the CO1 transcript. By contrast, we find that ELAC2 and PTCD1 affect the 3' end processing of tRNAs. Finally, we found that MRPP1 is essential for transcript processing, RNA modification, translation and mitochondrial respiration.
[show abstract][hide abstract] ABSTRACT: The cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and thioredoxins (Trx1 and Trx2) are key components of the mammalian thioredoxin system, which is important for antioxidant defense and redox regulation of cell function. TrxR1 and TrxR2 are selenoproteins generally considered to have comparable properties, but to be functionally separated by their different compartments. To compare their properties we expressed recombinant human TrxR1 and TrxR2 and determined their substrate specificities and inhibition by metal compounds. TrxR2 preferred its endogenous substrate Trx2 over Trx1, whereas TrxR1 efficiently reduced both Trx1 and Trx2. TrxR2 displayed strikingly lower activity with dithionitrobenzoic acid (DTNB), lipoamide, and the quinone substrate juglone compared to TrxR1, and TrxR2 could not reduce lipoic acid. However, Sec-deficient two-amino-acid-truncated TrxR2 was almost as efficient as full-length TrxR2 in the reduction of DTNB. We found that the gold(I) compound auranofin efficiently inhibited both full-length TrxR1 and TrxR2 and truncated TrxR2. In contrast, some newly synthesized gold(I) compounds and cisplatin inhibited only full-length TrxR1 or TrxR2 and not truncated TrxR2. Surprisingly, one gold(I) compound, [Au(d2pype)(2)]Cl, was a better inhibitor of TrxR1, whereas another, [(iPr(2)Im)(2)Au]Cl, mainly inhibited TrxR2. These compounds also inhibited TrxR activity in the cytoplasm and mitochondria of cells, but their cytotoxicity was not always dependent on the proapoptotic proteins Bax and Bak. In conclusion, this study reveals significant differences between human TrxR1 and TrxR2 in substrate specificity and metal compound inhibition in vitro and in cells, which may be exploited for development of specific TrxR1- or TrxR2-targeting drugs.
[show abstract][hide abstract] ABSTRACT: Although the basic components and mechanisms of mitochondrial transcription in mammals have been described, the components involved in mRNA processing, translation and stability remain largely unknown. In plants, pentatricopeptide domain RNA-binding proteins regulate the stability, expression and translation of mitochondrial transcripts; therefore, we investigated the role of an uncharacterized mammalian pentatricopeptide domain protein, (PTCD1), in mitochondrial RNA metabolism. We show that PTCD1 is a mitochondrial matrix protein which associates with leucine tRNAs and precursor RNAs that contain leucine tRNAs. Knockdown of PTCD1 in 143B osteosarcoma cells did not change mitochondrial mRNA levels; however, it increased the abundance precursor RNAs and of leucine tRNAs and PTCD1 overexpression led to a reduction of these RNAs. Lowering PTCD1 in cells increased levels of several mitochondria-encoded proteins and Complex IV activity, suggesting that PTCD1 acts as a negative regulator of leucine tRNA levels and hence mitochondrial translation.
Nucleic Acids Research 09/2009; 37(17):5859-67. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The basic components and mechanisms of mitochondrial transcription in mammals have been described, however, the components involved in mRNA processing, translation and stability remain largely unknown. In plants, pentatricopeptide domain RNA-binding proteins regulate the stability, expression and translation of mitochondrial transcripts. Here, we investigated the role of an uncharacterized mammalian pentatricopeptide domain protein, pentatricopeptide repeat domain protein 3 (PTCD3), and showed that it is a mitochondrial protein that associates with the small subunit of mitochondrial ribosomes. PTCD3 knockdown and over expression did not affect mitochondrial mRNA levels, suggesting that PTCD3 is not involved in RNA processing and stability. However, lowering PTCD3 in 143B osteosarcoma cells decreased mitochondrial protein synthesis, mitochondrial respiration and the activity of Complexes III and IV, suggesting that PTCD3 has a role in mitochondrial translation.