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Composition of the mitoribosome of Diplonema papillatum

ABSTRACT: Biosynthesis of mitochondrial genome-encoded proteins is carried out by the mitoribosome, a specialized apparatus that has evolved and diverged dramatically since its bacterial origin. Recent studies across various eukaryotes have demonstrated widespread structural and compositional diversity of mitoribosomes. We used affinity pulldown of four mitoribosomal proteins to carry out a detailed analysis of mitoribosomes in Diplonema papillatum, the type species of diplonemids, a widespread group of single-celled marine flagellates. Using as baits mitoribosomal proteins integrating at distinct sites and phases during subunit maturation also allowed us to sample populations of mitoribosome assembly intermediates.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Diplonema Papillatum

SUBMITTER: Marlene Oeffinger

LAB HEAD: Marlene Oeffinger

PROVIDER: PXD040176 | Pride | 2023-10-24

REPOSITORIES: Pride

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Miniature RNAs are embedded in an exceptionally protein-rich mitoribosome via an elaborate assembly pathway.

Valach Matus M Benz Corinna C Aguilar Lisbeth C LC Gahura Ondřej O Faktorová Drahomíra D Zíková Alena A Oeffinger Marlene M Burger Gertraud G Gray Michael W MW Lukeš Julius J

Nucleic acids research 20230701 12

The mitochondrial ribosome (mitoribosome) has diverged drastically from its evolutionary progenitor, the bacterial ribosome. Structural and compositional diversity is particularly striking in the phylum Euglenozoa, with an extraordinary protein gain in the mitoribosome of kinetoplastid protists. Here we report an even more complex mitoribosome in diplonemids, the sister-group of kinetoplastids. Affinity pulldown of mitoribosomal complexes from Diplonema papillatum, the diplonemid type species, d ...[more]

PMID: 37207340

Similar Datasets

Composition of the mitoribosome of Diplonema papillatum

Project description:Biosynthesis of mitochondrial genome-encoded proteins is carried out by the mitoribosome, a specialized apparatus that has evolved and diverged dramatically since its bacterial origin. Recent studies across various eukaryotes have demonstrated widespread structural and compositional diversity of mitoribosomes. We used affinity pulldown of four mitoribosomal proteins to carry out a detailed analysis of mitoribosomes in Diplonema papillatum, the type species of diplonemids, a widespread group of single-celled marine flagellates. Using as baits mitoribosomal proteins integrating at distinct sites and phases during subunit maturation also allowed us to sample populations of mitoribosome assembly intermediates.

2023-05-19 | PXD039927 | Pride

Composition of the mitoribosome of Diplonema papillatum

Project description:Biosynthesis of mitochondrial genome-encoded proteins is carried out by the mitoribosome, a specialized apparatus that has evolved and diverged dramatically since its bacterial origin. Recent studies across various eukaryotes have demonstrated widespread structural and compositional diversity of mitoribosomes. We used sucrose gradient centrifugation and Blue-Native PAGE to separate mitoribosomes of Diplonema papillatum, the type species of diplonemids, a widespread group of single-celled marine flagellates.

2023-05-19 | PXD039882 | Pride

GTPBP8 is essential for mitoribosome formation in human mitochondria

Project description:Mitochondrial gene expression relies on mitoribosomes to translate mitochondrial mRNAs. The biogenesis of mitoribosomes is an intricate process involving multiple assembly factors. Among these factors, GTP-binding proteins (GTPBPs) play crucial roles. In bacterial systems, numerous GTPBPs are required for ribosome subunit maturation, with EngB being a GTPBP involved in the ribosomal large subunit assembly. In this study, we focused on exploring the function of GTPBP8, the human homolog of EngB. We found that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. In the absence of GTPBP8, numerous mitoribosomal proteins from both subunits become destabilized, indicating GTPBP8's involvement in synchronized subunit assembly. Furthermore, structural analysis of mitoribosomes from GTPBP8 knock-out cells showed the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Our study highlights the crucial role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitoribosome biogenesis.

2023-09-13 | GSE242965 | GEO

GTPBP8 is essential for mitoribosome formation in human mitochondria

2024-04-11 | PXD045264 | Pride

GTPase driven maturation of the human mitoribosomal peptidyl transferase center

Project description:We combine genetic perturbation with cryo-electron microscopy (cryo-EM) to establish the mechanism of PTC maturation during human mitoribosome biogenesis. Cryo-EM structures of large mitoribosomal subunit (mtLSU) assembly intermediates purified from GTPBP6-deficient cells reveal that GTPBP5 acts in concert with the methyltransferase domain of the NSUN4-MTERF4 complex to facilitate PTC folding. Addition of recombinant GTPBP6 to these assembly intermediates provides the structural basis for subsequent GTPBP6-mediated late PTC maturation and suggests a sequential order of mtLSU biogenesis. Finally, cryo-EM analysis of 55S mitoribosomes treated with GTPBP6 explains how this protein can adopt a dual role in ribosome biogenesis and recycling. Taken together, these results provide the molecular basis for PTC maturation and establish a hierarchical model for late mitoribosome biogenesis.

2021-05-20 | PXD023502 | Pride

Distinct roles for human mtIF2 and mtIF3 in non-canonical mitochondrial translation.

Project description:The synthesis of mitochondrial OXPHOS complexes is central to cellular metabolism, yet many molecular details of mitochondrial translation remain elusive. It is commonly held view that translation initiation in human mitochondria proceeded in a manner similar to bacterial systems, with the mitoribosomal small subunit bound to the initiation factors, mtIF2 and mtIF3, along with initiator tRNA and an mRNA. However, unlike in bacteria, most human mitochondrial mRNAs lack 5′ leader sequences that can mediate small subunit binding, raising the question of how leaderless mRNAs are recognized by mitoribosomes. By using novel in vitro mitochondrial translation initiation assays, alongside biochemical and genetic characterization of cellular knockouts of mitochondrial translation factors, we describe unique features of translation initiation in human mitochondria. We show that in vitro, leaderless mRNA transcripts can be loaded directly onto assembled 55S mitoribosomes, but not onto the mitoribosomal small subunit (28S). In addition, we demonstrate that while mtIF2 is indispensable for mitochondrial translation, mtIF3 activity is not required for translation of leaderless mitochondrial transcripts but is essential for translation of ATP6 in the case of the bicistronic ATP8/ATP6 transcript. Our results confirm important evolutionary divergences of the mitochondrial translation system, and further our understanding of a process central to eukaryotic metabolism.

2022-12-01 | GSE156937 | GEO

Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly

Project description:Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including NSUN4-MTERF4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolyzable analog of GTP, suggesting a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. CRISPR/Cas9-mediated GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation, reduced monosome formation and elevated levels of certain mitoribosome assembly factors. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage maturation of the mt-LSU maturation.

2020-11-09 | PXD017384 | Pride

Using mitoribosomal profiling to investigate human mitochondrial translation

Project description:Human 143B.206 Rho+ cells (depleted mitochondrial DNA) and cytoplasts (derived from mitochondrial patient fibroblast containing C1624T mutation in mitochondrial tRNAVal ) were previously used to generated transmitochondrial cybrid lines. Hek293T cells,143B cells and cybrid cells were cultured in DMEM supplemented with 10% FCS in 5% CO2 at 37 °C. Cells were treated with Chloramphenicol (100 μg/ ml) for 15 min before harvest. Cells were then lysed and RNase I digested. Lysates were then loaded on a sucrose gradient (7%-47%). Fractions containing mito-monosomes were used to isolated RNA which were used to generate the library for deep sequencing. Mitoribosomal profiling were generated for both control (143B and Hek293T) and cybrid cells to reveal aspects of mitoribosome behaviour.

2017-12-01 | E-MTAB-6284 | biostudies-arrayexpress

C6orf203 is an RNA-binding protein involved in mitochondrial protein synthesis

Project description:In all biological systems, RNAs are associated with RNA-binding proteins (RBPs), forming complexes that control gene regulatory mechanisms, from RNA synthesis to decay. In mammalian mitochondria, post-transcriptional regulation of gene expression is conducted by mitochondrial RBPs (mt-RBPs) at various stages of mt-RNA metabolism, including polycistronic transcript production, its processing into individual transcripts, mt-RNA modifications, stability, translation, and degradation. To date, only a handful of mt-RBPs have been characterized. Here, we describe a putative human mitochondrial protein, C6orf203, that contains an S4-like domain - an evolutionarily conserved RNA-binding domain previously identified in proteins involved in translation. Our data show C6orf203 to bind highly-structured RNA in vitro and associate with the mitoribosomal large subunit in HEK293T cells. Knockout of C6orf203 leads to a decrease in mitochondrial translation and consequent OXPHOS deficiency, without affecting mitochondrial RNA levels. Although mitoribosome stability is not affected in C6orf203-depleted cells, mitoribosome profiling analysis revealed a global disruption of the association of mt-mRNAs with the mitoribosome, suggesting that C6orf203 may be required for the proper maturation and functioning of the mitoribosome. We therefore propose C6orf203 to be a novel RNA-binding protein involved in mitochondrial translation, expanding the repertoire of factors engaged in this process.

2019-08-10 | GSE133315 | GEO

Deficiency of mitoribosomal S10 protein affects translation and splicing in Arabidopsis mitochondria

Project description:The ribosome is not only a protein-making machine, but also a regulatory element in protein synthesis. This view is supported by our earlier data showing that Arabidopsis mitoribosomes altered due to the silencing of the nuclear RPS10 gene encoding mitochondrial ribosomal protein S10 differentially translate mitochondrial transcripts compared with the wild-type. Here, we used ribosome profiling to determine the contribution of transcriptional and translational control in the regulation of protein synthesis in rps10 mitochondria compared with the wild-type ones. Oxidative phosphorylation system proteins are preferentially synthesized in wild-type mitochondria but this feature is lost in the mutant. The rps10 mitoribosomes show slightly reduced translation efficiency of most respiration-related proteins and at the same time markedly more efficiently synthesize ribosomal proteins and MatR and TatC proteins. The mitoribosomes deficient in S10 protein protect shorter transcript fragments which exhibit a weaker 3-nucleotide periodicity compared with the wild-type. The decrease in the triplet periodicity is particularly drastic for genes containing introns. Notably, splicing is considerably less effective in the mutant, indicating an unexpected link between the deficiency of S10 and mitochondrial splicing. Thus, a shortage of the mitoribosomal S10 protein has wide-ranging consequences on mitochondrial gene expression.

2019-10-29 | GSE132441 | GEO

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