The roles of assembly factors in mammalian mitoribosome biogenesis.

As ancient bacterial endosymbionts of eukaryotic cells, mitochondria have retained their own circular DNA as well as protein translation system including mitochondrial ribosomes (mitoribosomes). In recent years, methodological advancements in cryoelectron microscopy and mass spectrometry have revealed the extent of the evolutionary divergence of mitoribosomes from their bacterial ancestors and their adaptation to the synthesis of 13 mitochondrial DNA encoded oxidative phosphorylation complex subunits. In addition to the structural data, the first assembly pathway maps of mitoribosomes have started to emerge and concomitantly also the assembly factors involved in this process to achieve fully translational competent particles. These transiently associated factors assist in the intricate assembly process of mitoribosomes by enhancing protein incorporation, ribosomal RNA folding and modification, and by blocking premature or non-native protein binding, for example. This review focuses on summarizing the current understanding of the known mammalian mitoribosome assembly factors and discussing their possible roles in the assembly of small or large mitoribosomal subunits.

Linear Density Sucrose Gradients to Study Mitoribosomal Biogenesis in Tissue-Specific Knockout Mice.

Like bacterial and cytoplasmic ribosomes, mitoribosomes are large ribonucleoprotein complexes with molecular weights in the range of several million Daltons. Traditionally, studying the assembly of such high molecular weight complexes is done using ultracentrifugation through linear density gradients, which remains the method of choice due to its versatility and superior resolving power in the high molecular weight range. Here, we present a protocol for the analysis of mitoribosomal assembly in heart mitochondrial extracts using linear density sucrose gradients that we have previously employed to characterize the essential role of different mitochondrial proteins in mitoribosomal biogenesis. This protocol details in a stepwise manner a typical mitoribosomal assembly analysis starting with isolation of mitochondria, preparation and ultracentrifugation of the gradients, fractionation and ending with SDS-PAGE, and immunoblotting of the gradient fractions. Even though we provide an example with heart mitochondria, this protocol can be directly applied to virtually all mouse tissues, as well as cultured cells, with little to no modifications.

Mitochondrial RNA granules are fluid condensates positioned by membrane dynamics.

Mitochondria contain the genetic information and expression machinery to produce essential respiratory chain proteins. Within the mitochondrial matrix, newly synthesized RNA, RNA processing proteins and mitoribosome assembly factors form punctate sub-compartments referred to as mitochondrial RNA granules (MRGs)1-3. Despite their proposed importance in regulating gene expression, the structural and dynamic properties of MRGs remain largely unknown. We investigated the internal architecture of MRGs using fluorescence super-resolution localization microscopy and correlative electron microscopy, and found that the MRG ultrastructure consists of compacted RNA embedded within a protein cloud. Using live-cell super-resolution structured illumination microscopy and fluorescence recovery after photobleaching, we reveal that MRGs rapidly exchange components and can undergo fusion, characteristic properties of fluid condensates4. Furthermore, MRGs associate with the inner mitochondrial membrane and their fusion coincides with mitochondrial remodelling. Inhibition of mitochondrial fission or fusion leads to an aberrant accumulation of MRGs into concentrated pockets, where they remain as distinct individual units despite their close apposition. Together, our findings reveal that MRGs are nanoscale fluid compartments, which are dispersed along mitochondria via membrane dynamics.

Structural basis of mitochondrial translation.

Translation of mitochondrial messenger RNA (mt-mRNA) is performed by distinct mitoribosomes comprising at least 36 mitochondria-specific proteins. How these mitoribosomal proteins assist in the binding of mt-mRNA and to what extent they are involved in the translocation of transfer RNA (mt-tRNA) is unclear. To visualize the process of translation in human mitochondria, we report ~3.0 Å resolution structure of the human mitoribosome, including the L7/L12 stalk, and eight structures of its functional complexes with mt-mRNA, mt-tRNAs, recycling factor and additional trans factors. The study reveals a transacting protein module LRPPRC-SLIRP that delivers mt-mRNA to the mitoribosomal small subunit through a dedicated platform formed by the mitochondria-specific protein mS39. Mitoribosomal proteins of the large subunit mL40, mL48, and mL64 coordinate translocation of mt-tRNA. The comparison between those structures shows dynamic interactions between the mitoribosome and its ligands, suggesting a sequential mechanism of conformational changes.

Long Noncoding RNA MRPL39 Inhibits Gastric Cancer Proliferation and Progression by Directly Targeting miR-130.

BACKGROUND: Gastric cancer (GC) is one of the most prevalent malignant tumors displaying both high incidence and mortality throughout much of the world. Recently, long noncoding RNAs (lncRNAs) have been implicated in the development and progression of GC. MATERIALS AND METHODS: In the present study, we investigated the biological function and molecular mechanisms of lncRNA MRPL39 in GC. RESULTS: We found that MRPL39 was significantly downregulated in GC tissues and cell lines and that its expression level was negatively associated with carcinoma size, tumor, lymph node, metastasis (TNM) stage, and lymphatic metastasis. Patients with low MRPL39 expression levels revealed a short overall and disease-free survival period. Over-expression of MRPL39 in the GC cell lines BGC823 and SGC-7901 inhibited cell growth, proliferation, migration, and invasion. MiR-130, a putative target gene of MRPL39, displayed an inverse association with the expression of MRPL39 in GC tissues and cell lines. Moreover, a luciferase assay demonstrated a direct binding between the miR-130 and MRPL39, and the reintroduction of miR-130 abrogated the anti-tumor effect of MRPL39 on GC cells. CONCLUSION: Taken together, these findings indicate that MRPL39 serves as a tumor suppressor by directly targeting miR-130 in GC, which suggests that it might be a novel biomarker in the diagnosis and prognosis of GC.

Fyn kinase regulates translation in mammalian mitochondria.

BACKGROUND: Mitochondrial translation machinery solely exists for the synthesis of 13 mitochondrially-encoded subunits of the oxidative phosphorylation (OXPHOS) complexes in mammals. Therefore, it plays a critical role in mitochondrial energy production. However, regulation of the mitochondrial translation machinery is still poorly understood. In comprehensive proteomics studies with normal and diseased tissues and cell lines, we and others have found the majority of mitochondrial ribosomal proteins (MRPs) to be phosphorylated. Neither the kinases for these phosphorylation events nor their specific roles in mitochondrial translation are known. METHODS: Mitochondrial kinases are responsible for phosphorylation of MRPs enriched from bovine mitoplasts by strong cation-exchange chromatography and identified by mass spectrometry-based proteomics analyses of kinase rich fractions. Phosphorylation of recombinant MRPs and 55S ribosomes was assessed by in vitro phosphorylation assays using the kinase-rich fractions. The effect of identified kinase on OXPHOS and mitochondrial translation was assessed by various cell biological and immunoblotting approaches. RESULTS: Here, we provide the first evidence for the association of Fyn kinase, a Src family kinase, with mitochondrial translation components and its involvement in phosphorylation of 55S ribosomal proteins in vitro. Modulation of Fyn expression in human cell lines has provided a link between mitochondrial translation and energy metabolism, which was evident by the changes in 13 mitochondrially encoded subunits of OXPHOS complexes. CONCLUSIONS AND GENERAL SIGNIFICANCE: Our findings suggest that Fyn kinase is part of a complex mechanism that regulates protein synthesis and OXPHOS possibly by tyrosine phosphorylation of translation components in mammalian mitochondria.

Proteomic profiling of the mitochondrial ribosome identifies Atp25 as a composite mitochondrial precursor protein.

Whereas the structure and function of cytosolic ribosomes are well characterized, we only have a limited understanding of the mitochondrial translation apparatus. Using SILAC-based proteomic profiling, we identified 13 proteins that cofractionated with the mitochondrial ribosome, most of which play a role in translation or ribosomal biogenesis. One of these proteins is a homologue of the bacterial ribosome-silencing factor (Rsf). This protein is generated from the composite precursor protein Atp25 upon internal cleavage by the matrix processing peptidase MPP, and in this respect, it differs from all other characterized mitochondrial proteins of baker's yeast. We observed that cytosolic expression of Rsf, but not of noncleaved Atp25 protein, is toxic. Our results suggest that eukaryotic cells face the challenge of avoiding negative interference from the biogenesis of their two distinct translation machineries.

The 55S mammalian mitochondrial ribosome and its tRNA-exit region.

Mitochondria carry their own genetic material and gene-expression machinery, including ribosomes, which are responsible for synthesizing polypeptides that form essential components of the complexes involved in oxidative phosphorylation (or ATP generation) for the eukaryotic cell. Mitochondrial ribosomes (mitoribosomes) are quite divergent from cytoplasmic ribosomes in both composition and structure even as their main functional cores, such as the mRNA decoding and peptidyl transferase sites, are highly conserved. Remarkable progress has been made recently towards understanding the structure of mitoribosomes, by obtaining high-resolution cryo-electron microscopic (cryo-EM) maps. These studies confirm previous structural findings that had revealed that a significant reduction in size of ribosomal RNAs has caused topological changes in some of the functionally relevant regions, including the transfer RNA (tRNA)-binding sites and the nascent polypeptide-exit tunnel, within the structure of the mammalian mitoribosome. In addition, these studies provide unprecedented detailed views of the molecular architecture of those regions. In this review, we summarize the current state of knowledge of the structure of the mammalian mitoribosome and describe the molecular environment of its tRNA-exit region.