Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

RNA Post-transcriptional Modifications of an Early-Stage Large Subunit Ribosomal Intermediate

ABSTRACT: Protein production by the ribosomes is fundamental to life and proper assembly of the ribosome is required for protein production. The RNA, which is post-transcriptionally modified, provides the platform for ribosome assembly. Thus, a complete understanding of ribosome assembly requires the determination of the RNA post-transcriptional modifications in all the ribosome assembly intermediates and on each pathway. There are 26 RNA post-transcriptional modifications in 23S RNA of the mature Escherichia coli (E.coli) large ribosomal subunit. The levels of these modifications have been investigated extensively only for a small number of large subunit intermediates and under a limited number of cellular and environmental conditions. In this study we determine the level of incorporations of 2-methyl adenosine, 3-methyl pseudouridine, 5-hydroxycytosine and eight pseudouridines in an early-stage E.coli large subunit assembly intermediate with a sedimentation coefficient of 27S. The 27S intermediate is one of three large subunit intermediates accumulated in E.coli cells lacking the DEAD-box RNA helicase DbpA and expressing the helicase inactive R331A DbpA construct. The majority of the investigated modifications are incorporated into the 27S large subunit intermediate to similar levels as in the mature 50S large subunit, indicating that these early modifications or the enzymes that incorporate them have important roles in the initial events of large subunit ribosome assembly.

ORGANISM(S): Escherichia coli

PROVIDER: GSE232539 | GEO | 2024/05/15

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Shared Molecules

Only show the datasets with similarity scores above: 0.5

Threshold

0.5

Similar Datasets

RNA Post-transcriptional Modifications in Two Large Subunit Intermediates Populated in E.coli Cells Expressing Helicase Inactive R331A DbpA

Project description:RNA Post-transcriptional Modifications in Two Large Subunit Intermediates Populated in E.coli Cells Expressing Helicase Inactive R331A DbpA

| PRJNA807363 | ENA

RNA Post-transcriptional Modifications in Two Large Subunit Intermediates Populated in E.coli Cells Expressing Helicase Inactive R331A DbpA

Project description:23S ribosomal RNA (rRNA) of Escherichia coli 50S large ribosome subunit contains 26 post-transcriptionally modified nucleosides. Here, we determine the extent of modifications in the 35S and 45S large subunit intermediates, accumulating in cells expressing the helicase inactive DbpA protein, R331A, and the native 50S large subunit. The modifications we characterized are nine pseudouridines, 3-methylpseudouridine, 2-methyladenine, and 5-hydroxycytosine. These modifications were detected using 1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT) treatment followed by alkaline treatment. In addition, KMnO4 treatment of 23S rRNA was employed to detect 5-hydroxycytosine modification. CMCT and KMnO4 treatment produce chemical changes in modified nucleotides that cause reverse transcriptase misincorporations and deletions, which were detected employing next generation sequencing. Our results show that seven uridines to pseudouridine isomerizations and the 2-methyladenosine modification are present both in the 35S and 45S to similar extents as in the 50S. Hence, the enzymes that perform these modifications, namely RluF, RluB, RlmN, RluA, RluC, and RluA, have already acted in the intermediates. While two uridines to pseudouridines isomerizations, the 3-methylpseudouridine, and 5-hydroxycytosine modifications are significantly less present in the 35S and 45S as compared to the 50S. Therefore, the enzymes that perform these modifications, RluD, RlmH, and RlhA, are in the process of modifying the 35S, and 45S or these enzymes act during the later stages of ribosome assembly. Our study employs a novel high throughput and single nucleotide resolution technique for detection of 2-methyladenine and two novel high throughput and single nucleotide resolution technique for detection of 5-hydroxycytosine.

2022-09-15 | GSE196821 | GEO

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. 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. Furthermore, fPAR-CLIP analysis revealed that GTPBP8 is an RNA-binding protein that interacts specifically with the mitochondrial ribosome large subunit16S rRNA. Our study highlights the crucial role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitochondrial large subunit maturation.

2024-06-19 | GSE256250 | GEO

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

RNA Post-transcriptional Modifications of an Early-Stage Large Subunit Ribosomal Intermediate

Project description:RNA Post-transcriptional Modifications of an Early-Stage Large Subunit Ribosomal Intermediate

| PRJNA972686 | ENA

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.

2024-04-11 | PXD045264 | Pride

The RNA helicase Dbp10 coordinates assembly factor association with PTC formation during ribosome biogenesis

Project description:During ribosome biogenesis a plethora of assembly factors and essential enzymes drive the unidirectional maturation of nascent pre-ribosomal subunits. The DEAD-box RNA helicase Dbp10 is suggested to restructure pre-ribosomal rRNA of the evolving peptidyl-transferase center (PTC) on nucleolar ribosomal 60S assembly intermediates. Here, we show that point mutations within conserved catalytic helicase-core motifs of Dbp10 yield a dominant-lethal growth phenotype. Such dbp10 mutants, which stably associate with pre-60S intermediates, impair pre-60S biogenesis at a nucleolar stage prior to the release of assembly factor Rrp14 and stable integration of late nucleolar factors such as Noc3. Furthermore, the binding of the GTPase Nug1 to particles isolated directly via mutant Dbp10 bait proteins is specifically inhibited. The N-terminal domain of Nug1 interacts with Dbp10 and the methyltransferase Spb1, whose pre-60S incorporation is also reduced in absence of functional Dbp10. Our data suggest that Dbp10’s helicase activity generates a crucial framework for assembly factor docking thereby permitting the progression of pre-60S maturation

2024-05-22 | PXD046068 | Pride

Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution

Project description:Ribosome assembly in eukaryotes involves the activity of hundreds of assembly factors that direct the hierarchical assembly of ribosomal proteins and numerous ribosomal RNA folding steps. However, detailed insights into the function of assembly factors and ribosomal RNA folding events are lacking. To address this, we have developed ChemModSeq, a method that combines structure probing, high throughput sequencing and statistical modeling, to quantitatively measure RNA structural rearrangements during the assembly of macromolecular complexes. By applying ChemModSeq to purified 40S assembly intermediates we obtained nucleotide-resolution maps of ribosomal RNA flexibility revealing structurally distinct assembly intermediates and mechanistic insights into assembly dynamics not readily observed in cryo-electron microscopy reconstructions. We show that RNA restructuring events coincide with the release of assembly factors and predict that completion of the head domain is required before the Rio1 kinase enters the assembly pathway. Collectively, our results suggest that 40S assembly factors regulate the timely incorporation of ribosomal proteins by delaying specific folding steps in the 3M-bM-^@M-^Y major domain of the 20S pre-ribosomal RNA. Three datasets of yeast ribosomal samples subjected to different chemical modifications; 1M7 dataset contains 8 different modified samples and 2 control samples; NAI dataset contains 3 different modified samples and 2 control samples; DMS dataset contains 1 modified sample and 1 control sample. Each sample consists of at least two replicates.

2014-11-14 | E-GEOD-52878 | biostudies-arrayexpress

UtpA and UtpB chaperone nascent pre-ribosomal RNA and the U3 snoRNP to initiate eukaryotic ribosome assembly

Project description:Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes – UtpA and UtpB – interact with nascent pre-ribosomal RNA have so far been poorly understood. We have combined biochemical and structural biology approaches with ensembles of RNA-protein cross-linking to elucidate the essential function of both complexes. Here we show that UtpA contains a large composite RNA binding site and captures the 5´ end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to key architectural sites such as an RNA duplex formed by the 5´ ETS and U3 snoRNA as well as the 3´ boundary of 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.

2016-09-21 | GSE79950 | GEO

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

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