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The discovery of a wild abortive-type (WA) cytoplasmic male sterile (CMS) line and breeding its restorer line have led to the commercialization of three-line hybrid rice, contributing considerably to global food security. However, the molecular mechanisms underlying fertility abortion and the restoration of CMS-WA lines remain largely elusive. In this study, we cloned a restorer gene, Rf20 , following a genome-wide association study analysis of the core parent lines of three-line hybrid rice. We found that Rf20 was present in all core parental lines, but different haplotypes and structural variants of its gene resulted in differences in Rf20 expression levels between sterile and restored lines. Rf20 could restore pollen fertility in the CMS-WA line and was found to be responsible for fertility restoration in some CMS lines under high temperatures. In addition, we found that Rf20 encodes a pentatricopeptide repeat protein that competes with WA352 for binding with COX11. This interaction enhances COX11's function as a scavenger of reactive oxygen species, which in turn restores pollen fertility. Collectively, our study suggests a new action mode for pentatricopeptide repeat proteins in the fertility restoration of CMS lines, providing an essential theoretical basis for breeding robust restorer lines and for overcoming high temperature-induced fertility recovery of some CMS lines. Cytoplasmic male sterility (CMS) is an important genetic tool for breeding and exploiting hybrid vigor in crops. Rf genes can make CMS lines fertile. Song et al. show that structural variations of a new fertility restorer gene, Rf20 , underlie the restoration of wild abortive-type CMS in rice. Mechanistically, RF20 can compete with WA352 for binding with COX11 to enhance the scavenging of reactive oxygen species for the fertility restoration. [ABSTRACT FROM AUTHOR]

Primary mitochondrial diseases are a group of genetically and clinically heterogeneous disorders resulting from oxidative phosphorylation (OXPHOS) defects. COX11 encodes a copper chaperone that participates in the assembly of complex IV and has not been previously linked to human disease. In a previous study, we identified that COX11 knockdown decreased cellular adenosine triphosphate (ATP) derived from respiration, and that ATP levels could be restored with coenzyme Q10 (CoQ10 ) supplementation. This finding is surprising since COX11 has no known role in CoQ10 biosynthesis. Here, we report a novel gene-disease association by identifying biallelic pathogenic variants in COX11 associated with infantile-onset mitochondrial encephalopathies in two unrelated families using trio genome and exome sequencing. Functional studies showed that mutant COX11 fibroblasts had decreased ATP levels which could be rescued by CoQ10 . These results not only suggest that COX11 variants cause defects in energy production but reveal a potential metabolic therapeutic strategy for patients with COX11 variants.

Introduction: Long noncoding RNAs (lncRNAs) have been implicated in the pathogenesis of allergic rhinitis (AR). The current investigation is focused on elucidating the functional impact of a specific lncRNA, FGD5 antisense RNA 1 (FGD5-AS1), on the development and progression of AR through its interaction with miR-223-3p. Methods: An experimental framework for AR was constructed in both cellular and animal models. Quantitative assessment of FGD5-AS1, miR-223-3p, and COX11 mRNA expression was conducted using real-time quantitative reverse transcription PCR. The expression of inflammatory factors, immunoglobulin E, LTC4, and ECP, was examined using ELISA. Apoptosis in human nasal epithelial cells was assessed by the flow cytometry method. The protein expression of COX11 was examined using Western blotting. Nasal mucosal function was further evaluated by hematoxylin and eosin staining. Furthermore, bioinformatics evaluations, dual-luciferase reporter assays, and a series of experimental procedures unveiled a putative competitive endogenous RNA regulatory mechanism. Results: We found the expression of lncRNA FGD5-AS1 was decreased in AR. In vitro lncRNA FGD5-AS1 attenuated the production of inflammatory cytokines in nasal epithelial cells. Furthermore, elevated FGD5-AS1 expression significantly alleviated AR symptoms by reducing nasal epithelial apoptosis and inflammation. MiR-223-3p was identified as a direct target of FGD5-AS1. Moreover, miRNA-223-3p directly downregulated the expression of COX11 mRNA. Subsequent experiments confirmed that FGD5-AS1 regulated AR through the miR-223-3p/COX11 axis, thereby inhibiting inflammation. Conclusion: The FGD5-AS1/miR-223-3p/COX11 axis plays a pivotal role in the pathogenesis of AR, suggesting that FGD5-AS1 could serve as a potential diagnostic biomarker and therapeutic target for AR. [ABSTRACT FROM AUTHOR]

Abstract Background Copper is an essential metal for living organisms as a catalytic co-factor for important enzymes, like cytochrome c oxidase the final enzyme in the electron transport chain. Plasmodium falciparum parasites in infected red blood cells are killed by excess copper and development in erythrocytes is inhibited by copper chelators. Cytochrome c oxidase in yeast obtains copper for the CuB site in the Cox1 subunit from Cox11. Methods A 162 amino acid carboxy-terminal domain of the P. falciparum Cox11 ortholog (PfCox11Ct) was recombinantly expressed and the rMBPPfCox11Ct affinity purified. Copper binding was measured in vitro and in Escherichia coli host cells. Site directed mutagenesis was used to identify key copper binding cysteines. Antibodies confirmed the expression of the native protein. Results rMBPPfCox11Ct was expressed as a 62 kDa protein fused with the maltose binding protein and affinity purified. rMBPPfCox11Ct bound copper measured by: a bicinchoninic acid release assay; atomic absorption spectroscopy; a bacterial host growth inhibition assay; ascorbate oxidation inhibition and in a thermal shift assay. The cysteine 157 amino acid was shown to be important for in vitro copper binding by PfCox11whilst Cys 60 was not. The native protein was detected by antibodies against rMBPPfCox11Ct. Conclusions Plasmodium spp. express the PfCox11 protein which shares structural features and copper binding motifs with Cox11 from other species. PfCox11 binds copper and is, therefore, predicted to transfer copper to the CuB site of Plasmodium cytochrome c oxidase. Characterization of Plasmodium spp. proteins involved in copper metabolism will help sceintists understand the role of cytochrome c oxidase and this essential metal in Plasmodium homeostasis.

The mitochondrial genomes are very diverse, but their evolutionary history is unclear due to the lack of efforts to sequence those of protists (unicellular eukaryotes), which cover a major part of the eukaryotic tree. Cryptista comprises cryptophytes, goniomonads, kathablepharids, and Palpitomonas bilix, and their mitochondrial genomes (mt-genomes) are characterized by various gene contents, particularly the presence/absence of an ancestral (bacterial) system for the cytochrome c maturation system. To shed light on mt-genome evolution in Cryptista, we report the complete mt-genome of Microheliella maris, which was recently revealed to branch at the root of Cryptista. The M. maris mt-genome was reconstructed as a circular mapping chromosome of 61.2 kbp with a pair of inverted repeats (12.9 kbp) and appeared to be the most gene-rich among the mt-genomes of the members of Diaphoretickes (a mega-scale eukaryotic assembly including Archaeplastida, Cryptista, Haptista, and SAR) studied so far, carrying 53 protein-coding genes. With this newly sequenced mt-genome, we inferred and discussed the evolution of the mt-genome in Cryptista and Diaphoretickes.

Cytochrome c oxidase (CcO) is a multimeric copper-containing enzyme of the mitochondrial respiratory chain that powers cellular energy production. The two core subunits of cytochrome c oxidase, Cox1 and Cox2, harbor the catalytic CuB and CuA sites, respectively. Biogenesis of each copper site occurs separately and requires multiple proteins that constitute the mitochondrial copper delivery pathway. Currently, the identity of all the members of the pathway is not known, though several evolutionarily conserved twin CX9Cmotif-containing proteins have been implicated in this process. Here, we performed a targeted yeast suppressor screen that placed Coa4, a twin CX9C motif-containing protein, in the copper delivery pathway to the Cox1 subunit. Specifically, we show that overexpression of Cox11, a copper metallochaperone required for the formation of CuB site, can restore Cox1 abundance, cytochrome c oxidase assembly, and mitochondrial respiration in coa4D cells. This rescue is dependent on the copper-coordinating cysteines of Cox11. The abundance of Coa4 and Cox11 in mitochondria is reciprocally regulated, further linking Coa4 to the CuB site biogenesis. Additionally, we find that coa4D cells have reduced levels of copper and exogenous copper supplementation can partially ameliorate its respiratory-deficient phenotype, a finding that connects Coa4 to cellular copper homeostasis. Finally, we demonstrate that human COA4 can replace the function of yeast Coa4 indicating its evolutionarily conserved role. Our work provides genetic evidences for the role of Coa4 in the copper delivery pathway to the CuB site of cytochrome c oxidase. [ABSTRACT FROM AUTHOR]

Background: Copper is an essential metal for living organisms as a catalytic co-factor for important enzymes, like cytochrome c oxidase the final enzyme in the electron transport chain. Plasmodium falciparum parasites in infected red blood cells are killed by excess copper and development in erythrocytes is inhibited by copper chelators. Cytochrome c oxidase in yeast obtains copper for the CuB site in the Cox1 subunit from Cox11. Methods: A 162 amino acid carboxy-terminal domain of the P. falciparum Cox11 ortholog (PfCox11Ct) was recombinantly expressed and the rMBPPfCox11Ct affinity purified. Copper binding was measured in vitro and in Escherichia coli host cells. Site directed mutagenesis was used to identify key copper binding cysteines. Antibodies confirmed the expression of the native protein. Results: rMBPPfCox11Ct was expressed as a 62 kDa protein fused with the maltose binding protein and affinity purified. rMBPPfCox11Ct bound copper measured by: a bicinchoninic acid release assay; atomic absorption spectroscopy; a bacterial host growth inhibition assay; ascorbate oxidation inhibition and in a thermal shift assay. The cysteine 157 amino acid was shown to be important for in vitro copper binding by PfCox11whilst Cys 60 was not. The native protein was detected by antibodies against rMBPPfCox11Ct. Conclusions: Plasmodium spp. express the PfCox11 protein which shares structural features and copper binding motifs with Cox11 from other species. PfCox11 binds copper and is, therefore, predicted to transfer copper to the CuB site of Plasmodium cytochrome c oxidase. Characterization of Plasmodium spp. proteins involved in copper metabolism will help sceintists understand the role of cytochrome c oxidase and this essential metal in Plasmodium homeostasis. [ABSTRACT FROM AUTHOR]

Plant cytoplasmic male sterility (CMS) is an important phenomenon and is widely utilized in hybrid crop breeding. The Wild Abortive CMS (CMS-WA), a well-known CMS type, has been successfully applied in the commercial production of hybrid rice seeds for more than 40 years. The CMS-WA causal gene WA352 encodes a novel transmenbrane protein and the interacts with the mitochondrial copper chaperone COX11, triggering reactive oxygen species production and resulting in male sterility in CMS-WA lines. However, the structure of WA352 is currently unknown, and the structural mechanism whereby WA352 perturbs COX11 function to cause CMS remains largely unknown. Here, we report the crystal structure of the C-terminal functional domain of WA352 at 1.3 Å resolution. This functional domain, consisting of five α helices, is spindle-shaped with a length of 42 Å, and a diameter of 28 Å. Notably, the absence of any structural similarity to a known protein structure suggests that the WA352 functional domain is a novel fold. In addition, surface conservation analysis and structural modeling of the WA352-COX11 complex revealed details about the WA352-COX11 interaction. Further structural analysis suggested that the WA352-COX11 interaction blocks the copper ion transportation activity of COX11, which is essential for the assembly of cytochrome c oxidase, resulting in male sterility in CMS-WA lines. Our study paves the way toward structural determination of the WA352-COX11 complex and provides new insight into the mechanism of plant CMS. [ABSTRACT FROM AUTHOR]

Abstract: Biogenesis of cytochrome c oxidase is a complex process involving more than 30 known accessory proteins in yeast for the regulation of transcription and translation, membrane insertion and protein processing, cofactor insertion, and subunit assembly. Here, we focus on the process of cofactor insertion into subunit I of cytochrome c oxidase using the soil bacterium Paracoccus denitrificans as a model organism. The use of bacterial systems facilitates biogenesis studies, as the number of required assembly factors is reduced to a minimum. Both, co- and posttranslational cofactor insertion scenarios are discussed, and several approaches to shed light on this aspect of biogenesis are presented. CtaG, the Paracoccus homolog of yeast Cox11 which is involved in copper delivery to the CuB center, has been purified and characterized spectroscopically. A previously unreported signal at 358 nm allows monitoring copper transfer from copper-loaded CtaG to an acceptor. Both CtaG and apo-subunit I were purified after expression in Escherichia coli to develop an in vitro copper transfer system, probing the posttranslational insertion hypothesis. To mimic a potential cotranslational insertion process, cell-free expression systems using E. coli and P. denitrificans extracts have been established. Expression of subunit I in the presence of the detergent Brij-35 produces high amounts of “solubilized” subunit I which can be purified in good yield. With this system it may be feasible to trap and purify assembly intermediates after adding free cofactors, purified assembly proteins, or P. denitrificans membranes. [Copyright &y& Elsevier]