Browse
Submit Data
Databases
API
Help

Dataset Information

26 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A distinct assembly pathway of the human 39S late pre-mitoribosome.

ABSTRACT: Assembly of the mitoribosome is largely enigmatic and involves numerous assembly factors. Little is known about their function and the architectural transitions of the pre-ribosomal intermediates. Here, we solve cryo-EM structures of the human 39S large subunit pre-ribosomes, representing five distinct late states. Besides the MALSU1 complex used as bait for affinity purification, we identify several assembly factors, including the DDX28 helicase, MRM3, GTPBP10 and the NSUN4-mTERF4 complex, all of which keep the 16S rRNA in immature conformations. The late transitions mainly involve rRNA domains IV and V, which form the central protuberance, the intersubunit side and the peptidyltransferase center of the 39S subunit. Unexpectedly, we find deacylated tRNA in the ribosomal E-site, suggesting a role in 39S assembly. Taken together, our study provides an architectural inventory of the distinct late assembly phase of the human 39S mitoribosome.

SUBMITTER: Cheng J

PROVIDER: S-EPMC8316566 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

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 MTERF4:NSUN4 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-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.

| S-EPMC7797037 | biostudies-literature

Systematic Analysis of Assembly Intermediates in Yeast to Decipher the Mitoribosome Assembly Pathway.

Project description:Studies of yeast mitoribosome assembly have been historically hampered by the difficulty of generating mitoribosome protein-coding gene deletion strains with a stable mitochondrial genome. The identification of mitochondrial DNA-stabilizing approaches allows for the generation of a complete set of yeast deletion strains covering all mitoribosome proteins and known assembly factors. These strains can be used to analyze the integrity and assembly state of mitoribosomes by determining the sedimentation profile of these structures by sucrose gradient centrifugation of mitochondrial extracts, coupled to mass spectrometry analysis of mitoribosome composition. Subsequent hierarchical cluster analysis of mitoribosome subassemblies accumulated in mutant strains reveals details regarding the order of protein association during the mitoribosome biogenetic process. These strains also allow the expression of truncated protein variants to probe the role of mitochondrion-specific protein extensions, the relevance of protein cofactors, or the importance of RNA-protein interactions in functional sites of the mitoribosome. In this chapter, we will detail the methodology involved in these studies.

| S-EPMC10654547 | biostudies-literature

Distinct mechanisms of the human mitoribosome recycling and antibiotic resistance.

Project description:Ribosomes are recycled for a new round of translation initiation by dissociation of ribosomal subunits, messenger RNA and transfer RNA from their translational post-termination complex. Here we present cryo-EM structures of the human 55S mitochondrial ribosome (mitoribosome) and the mitoribosomal large 39S subunit in complex with mitoribosome recycling factor (RRFmt) and a recycling-specific homolog of elongation factor G (EF-G2mt). These structures clarify an unusual role of a mitochondria-specific segment of RRFmt, identify the structural distinctions that confer functional specificity to EF-G2mt, and show that the deacylated tRNA remains with the dissociated 39S subunit, suggesting a distinct sequence of events in mitoribosome recycling. Furthermore, biochemical and structural analyses reveal that the molecular mechanism of antibiotic fusidic acid resistance for EF-G2mt is markedly different from that of mitochondrial elongation factor EF-G1mt, suggesting that the two human EF-Gmts have evolved diversely to negate the effect of a bacterial antibiotic.

| S-EPMC8203779 | biostudies-literature

Distinct mechanisms of the human mitoribosome recycling and antibiotic resistance

Project description: Not available

| EMPIAR-10703 | biostudies-other

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

Role of GTPases in Driving Mitoribosome Assembly.

Project description:Mitoribosomes catalyze essential protein synthesis within mitochondria. Mitoribosome biogenesis is assisted by an increasing number of assembly factors, among which guanosine triphosphate hydrolases (GTPases) are the most abundant class. Here, we review recent progress in our understanding of mitoribosome assembly GTPases. We describe their shared and specific features and mechanisms of action, compare them with their bacterial counterparts, and discuss their possible roles in the assembly of small or large mitoribosomal subunits and the formation of the monosome by establishing quality-control checkpoints during these processes. Furthermore, following the recent unification of the nomenclature for the mitoribosomal proteins, we also propose a unified nomenclature for mitoribosome assembly GTPases.

| S-EPMC7954922 | biostudies-literature

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

Project description: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, demonstrates that they have a mass of > 5 MDa, contain as many as 130 integral proteins, and exhibit a protein-to-RNA ratio of 11:1. This unusual composition reflects unprecedented structural reduction of ribosomal RNAs, increased size of canonical mitoribosomal proteins, and accretion of three dozen lineage-specific components. In addition, we identified >50 candidate assembly factors, around half of which contribute to early mitoribosome maturation steps. Because little is known about early assembly stages even in model organisms, our investigation of the diplonemid mitoribosome illuminates this process. Together, our results provide a foundation for understanding how runaway evolutionary divergence shapes both biogenesis and function of a complex molecular machine.

| S-EPMC10325924 | biostudies-literature

The Human Mitochondrial DEAD-Box Protein DDX28 Resides in RNA Granules and Functions in Mitoribosome Assembly.

Project description:Human mitochondrial ribosomes are specialized in the synthesis of 13 proteins, which are fundamental components of the oxidative phosphorylation system. The pathway of mitoribosome biogenesis, the compartmentalization of the process, and factors involved remain largely unknown. Here, we have identified the DEAD-box protein DDX28 as an RNA granule component essential for the biogenesis of the mitoribosome large subunit (mt-LSU). DDX28 interacts with the 16S rRNA and the mt-LSU. RNAi-mediated DDX28 silencing in HEK293T cells does not affect mitochondrial mRNA stability or 16S rRNA processing or modification. However, it leads to reduced levels of 16S rRNA and mt-LSU proteins, impaired mt-LSU assembly, deeply attenuated mitochondrial protein synthesis, and consequent failure to assemble oxidative phosphorylation complexes. Our findings identify DDX28 as essential during the early stages of mitoribosome mt-LSU biogenesis, a process that takes place mainly near the mitochondrial nucleoids, in the compartment defined by the RNA granules.

| S-EPMC4534351 | biostudies-literature

MTG1 couples mitoribosome large subunit assembly with intersubunit bridge formation.

Project description:Mammalian mitochondrial ribosomes (mitoribosomes) synthesize 13 proteins, essential components of the oxidative phosphorylation system. They are linked to mitochondrial disorders, often involving cardiomyopathy. Mitoribosome biogenesis is assisted by multiple cofactors whose specific functions remain largely uncharacterized. Here, we examined the role of human MTG1, a conserved ribosome assembly guanosine triphosphatase. MTG1-silencing in human cardiomyocytes and developing zebrafish revealed early cardiovascular lesions. A combination of gene-editing and biochemical approaches using HEK293T cells demonstrated that MTG1 binds to the large subunit (mtLSU) 16S ribosomal RNA to facilitate incorporation of late-assembly proteins. Furthermore, MTG1 interacts with mtLSU uL19 protein and mtSSU mS27, a putative guanosine triphosphate-exchange factor (GEF), to enable MTG1 release and the formation of the mB6 intersubunit bridge. In this way, MTG1 establishes a quality control checkpoint in mitoribosome assembly. In conclusion, MTG1 controls mitochondrial translation by coupling mtLSU assembly with intersubunit bridge formation using the intrinsic GEF activity acquired by the mtSSU through mS27, a unique occurrence in translational systems.

| S-EPMC6144824 | biostudies-literature

Human GTPBP10 is required for mitoribosome maturation.

Project description:Most steps on the biogenesis of the mitochondrial ribosome (mitoribosome) occur near the mitochondrial DNA nucleoid, in RNA granules, which contain dedicated RNA metabolism and mitoribosome assembly factors. Here, analysis of the RNA granule proteome identified the presence of a set of small GTPases that belong to conserved families of ribosome assembly factors. We show that GTPBP10, a member of the conserved Obg family of P-loop small G proteins, is a mitochondrial protein and have used gene-editing technologies to create a HEK293T cell line KO for GTPBP10. The absence of GTPBP10 leads to attenuated mtLSU and mtSSU levels and the virtual absence of the 55S monosome, which entirely prevents mitochondrial protein synthesis. We show that a fraction of GTPBP10 cosediments with the large mitoribosome subunit and the monosome. GTPBP10 physically interacts with the 16S rRNA, but not with the 12S rRNA, and crosslinks with several mtLSU proteins. Additionally, GTPBP10 is indirectly required for efficient processing of the 12S-16S rRNA precursor transcript, which could explain the mtSSU accumulation defect. We propose that GTPBP10 primarily ensures proper mtLSU maturation and ultimately serves to coordinate mtSSU and mtLSU accumulation then providing a quality control check-point function during mtLSU assembly that minimizes premature subunit joining.

| S-EPMC6265488 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data