GTPBP8 is required for mitoribosomal biogenesis and mitochondrial translation.

Mitochondrial translation occurs on the mitochondrial ribosome, also known as the mitoribosome. The assembly of mitoribosomes is a highly coordinated process. During mitoribosome biogenesis, various assembly factors transiently associate with the nascent ribosome, facilitating the accurate and efficient construction of the mitoribosome. However, the specific factors involved in the assembly process, the precise mechanisms, and the cellular compartments involved in this vital process are not yet fully understood. In this study, we discovered a crucial role for GTP-binding protein 8 (GTPBP8) in the assembly of the mitoribosomal large subunit (mt-LSU) and mitochondrial translation. GTPBP8 is identified as a novel GTPase located in the matrix and peripherally bound to the inner mitochondrial membrane. Importantly, GTPBP8 is specifically associated with the mt-LSU during its assembly. Depletion of GTPBP8 leads to an abnormal accumulation of mt-LSU, indicating that GTPBP8 is critical for proper mt-LSU assembly. Furthermore, the absence of GTPBP8 results in reduced levels of fully assembled 55S monosomes. This impaired assembly leads to compromised mitochondrial translation and, consequently, impaired mitochondrial function. The identification of GTPBP8 as an important player in these processes provides new insights into the molecular mechanisms underlying mitochondrial protein synthesis and its regulation.

Phenotype of Mrps5-Associated Phylogenetic Polymorphisms Is Intimately Linked to Mitoribosomal Misreading.

We have recently identified point mutation V336Y in mitoribosomal protein Mrps5 (uS5m) as a mitoribosomal ram (ribosomal ambiguity) mutation conferring error-prone mitochondrial protein synthesis. In vivo in transgenic knock-in animals, homologous mutation V338Y was associated with a discrete phenotype including impaired mitochondrial function, anxiety-related behavioral alterations, enhanced susceptibility to noise-induced hearing damage, and accelerated metabolic aging in muscle. To challenge the postulated link between Mrps5 V338Y-mediated misreading and the in vivo phenotype, we introduced mutation G315R into the mouse Mrps5 gene as Mrps5 G315R is homologous to the established bacterial ram mutation RpsE (uS5) G104R. However, in contrast to bacterial translation, the homologous G → R mutation in mitoribosomal Mrps5 did not affect the accuracy of mitochondrial protein synthesis. Importantly, in the absence of mitochondrial misreading, homozygous mutant MrpS5G315R/G315R mice did not show a phenotype distinct from wild-type animals.

Mitochondrial Ribosomal Protein L14 Promotes Cell Growth and Invasion by Modulating Reactive Oxygen Species in Thyroid Cancer.

OBJECTIVES: The mitochondrial ribosomal protein L14 (MRPL14) is encoded by a nuclear gene and participates in mitochondrial protein translation. In this study, we aimed to investigate the role of MRPL14 in thyroid cancer. METHODS: We investigated the association between MRPL14 expression and clinicopathological features using The Cancer Genome Atlas (TCGA) and Chungnam National University Hospital (CNUH) databases. Functional studies of MRPL14, including proliferation, migration, invasion, mitochondrial oxidative phosphorylation and reactive oxygen species (ROS) production, were performed in papillary thyroid cancer (PTC) cell lines (B-CPAP and KTC-1). RESULTS: Based on the TCGA dataset, PTC tissues lost mitochondrial integrity and showed dysregulated expression of overall mitoribosomal proteins (MRPs) compared with normal thyroid tissues. Of 78 MRPs, MRPL14 was highly expressed in thyroid cancer tissues. MRPL14 overexpression was significantly associated with advanced tumor stage, extrathyroidal extension, and lymph node metastasis. MRPL14 increased cell proliferation of thyroid cancer and promoted cell migration via epithelial-mesenchymal transition-related proteins. Moreover, MRPL14 knockdown reduced the expression of oxidative phosphorylation complex IV (MTCO1) and increased the accumulation of ROS. Cotreatment with a ROS scavenger restored cell proliferation and migration, which had been reduced by MRPL14 knockdown, implying that ROS functions as a key regulator of the oncogenic effects of MRPL14 in thyroid cancer cells. CONCLUSION: Our findings indicate that MRPL14 may promote cell growth, migration, and invasion by modulating ROS in thyroid cancer cells.

Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast.

The biogenesis of mitoribosomes is an intricate process that relies on the coordinated synthesis of nuclear-encoded mitoribosomal proteins (MRPs) in the cytosol, their translocation across mitochondrial membranes, the transcription of rRNA molecules in the matrix as well as the assembly of the roughly 80 different constituents of the mitoribosome. Numerous chaperones, translocases, processing peptidases, and assembly factors of the cytosol and in mitochondria support this complex reaction. The budding yeast Saccharomyces cerevisiae served as a powerful model organism to unravel the different steps by which MRPs are imported into mitochondria, fold into their native structures, and assemble into functional ribosomes.In this chapter, we provide established protocols to study these different processes experimentally. In particular, we describe methods to purify mitochondria from yeast cells, to import radiolabeled MRPs into isolated mitochondria, and to elucidate the assembly reaction of MRPs by immunoprecipitation. These protocols and the list of dos and don'ts will enable beginners and experienced scientists to study the import and assembly of MRPs.

Silencing of the mitochondrial ribosomal protein L-24 gene activates the oxidative stress response in Caenorhabditis elegans.

The mitochondrial translation machinery allows the synthesis of the mitochondrial-encoded subunits of the electron transport chain. Defects in this process lead to mitochondrial physiology failure; in humans, they are associated with early-onset, extremely variable and often fatal disorder. The use of a simple model to study the mitoribosomal defects is mandatory to overcome the difficulty to analyze the impact of pathological mutations in humans. In this paper we study in nematode Caenorhabditis elegans the silencing effect of the mrpl-24 gene, coding for the mitochondrial ribosomal protein L-24 (MRPL-24). This is a structural protein of the large subunit 39S of the mitoribosome and its effective physiological function is not completely elucidated. We have evaluated the nematode's fitness fault and investigated the mitochondrial defects associated with MRPL-24 depletion. The oxidative stress response activation due to the mitochondrial alteration has been also investigated as a compensatory physiological mechanism. For the first time, we demonstrated that MRPL-24 reduction increases the expression of detoxifying enzymes such as SOD-3 and GST-4 through the involvement of transcription factor SKN-1. BACKGROUND: In humans, mutations in genes encoding mitochondrial ribosomal proteins (MRPs) often cause early-onset, severe, fatal and extremely variable clinical defects. Mitochondrial ribosomal protein L-24 (MRPL24) is a structural protein of the large subunit 39S of the mitoribosome. It is highly conserved in different species and its effective physiological function is not completely elucidated. METHODS: We characterized the MRPL24 functionality using the animal model Caenorhabditis elegans. We performed the RNA mediated interference (RNAi) by exposing the nematodes' embryos to double-stranded RNA (dsRNA) specific for the MRPL-24 coding sequence. We investigated for the first time in C. elegans, the involvement of the MRPL-24 on the nematode's fitness and its mitochondrial physiology. RESULTS: Mrpl-24 silencing in C. elegans negatively affected the larval development, progeny production and body bending. The analysis of mitochondrial functionality revealed loss of mitochondrial network and impairment of mitochondrial functionality, as the decrease of oxygen consumption rate and the ROS production, as well as reduction of mitochondrial protein synthesis. Finally, the MRPL-24 depletion activated the oxidative stress response, increasing the expression levels of two detoxifying enzymes, SOD-3 and GST-4. CONCLUSIONS: In C. elegans the MRPL-24 depletion activated the oxidative stress response. This appears as a compensatory mechanism to the alteration of the mitochondrial functionality and requires the involvement of transcription factor SKN-1. GENERAL SIGNIFICANCE: C. elegans resulted in a good model for the study of mitochondrial disorders and its use as a simple and pluricellular organism could open interesting perspectives to better investigate the pathologic mechanisms underlying these devastating diseases.