RNA Post-transcriptional Modifications of an Early-Stage Large Subunit Ribosomal Intermediate
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA972686 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24564287_1.fastq.gz | Fastqsanger.gz | |||
| SRR24564287_2.fastq.gz | Fastqsanger.gz | |||
| SRR24564288_1.fastq.gz | Fastqsanger.gz | |||
| SRR24564288_2.fastq.gz | Fastqsanger.gz | |||
| SRR24564289_1.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 6 |
Similar Datasets
Project description: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.
2024-05-15 | GSE232539 | GEO
Project description:Yeast large ribosomal subunit (LSU) precursors are subject to substantial changes in protein composition during their maturation due to coordinated transient interactions with a large number of ribosome biogenesis factors and due to the assembly of ribosomal proteins. These compositional changes go along with stepwise processing of LSU rRNA precursors and with specific rRNA folding events, as revealed by recent cryo-electron microscopy analyses of late nuclear and cytoplasmic LSU precursors. Here we aimed to analyze changes in the spatial rRNA surrounding of selected ribosomal proteins during yeast LSU maturation. For this we combined a recently developed tethered tertiary structure probing approach with both targeted and high-throughput readout strategies. Several structural features of late LSU precursors were faithfully detected by this procedure. In addition, the obtained data let us suggest that early rRNA precursor processing events are accompanied by a global transition from a flexible to a spatially restricted rRNA conformation. For intermediate LSU precursors, a number of structural hallmarks could be addressed which include the fold of the internal transcribed spacer between 5.8S rRNA and 25S rRNA, the orientation of the central protuberance and the spatial organization of the interface between LSU rRNA domains I and III.
2017-04-01 | GSE97276 | GEO
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
Project description:The eukaryotic ribosome is a complex molecular machine responsible for the translation of mRNA to protein. Faulty ribosome production leads to decreased protein production, increased cellular stress, and often cell death. Previous research on ribosome biogenesis primarily focused on factors facilitating the proper assembly of the ribosome. By contrast, investigations into quality control mechanisms targeting abnormal pre-ribosomes remain relatively unexplored. We established a method to impede the maturation of a small subset of ribosomes in human cells. The assembly of the 60S large ribosomal subunit begins in the nucleolus, continues through the nucleoplasm, and concludes in the cytoplasm with the incorporation of the ribosomal protein uL16/RPL10 and the release of the late-acting biogenesis factors. We designed a system where incorporation of a mutant uL16 lacking residues 102-111 blocks the assembly of the maturing large subunit. To confirm that uL16mut incorporation blocks 60S maturation, we purified tagged uL16wt or uL16mut and identified associated proteins via western blotting and mass spectrometry analysis. We did not detect an interaction between uL16mut-containing large and subunits small subunit proteins, validating that the 60Smut subunits are trapped during biogenesis and unable to pair with small subunits to engage in active translation. We observed significant enrichment of late ribosome biogenesis factors, such as eIF6 and NMD3, upon uL16mut immunoprecipitation.
2025-04-28 | PXD051646 | Pride
Project description:These are the ribosomal subunit fractions from the polysome gradients. investigating effect of heat shock on procyclic-form trypanosomes.
2022-01-22 | E-MTAB-11314 | biostudies-arrayexpress
Project description:Structural basis of ribosomal 30S subunit degradation by RNase R
| PRJNA1055080 | ENA
Project description:The integrated stress response (ISR) is a central signalling network that enables eukaryotic cells to respond to a variety of different environmental stresses. Such stresses cause ribosome collisions that lead to activation of the kinase Gcn2, resulting in the phosphorylation and inactivation of eukaryotic initiation factor 2 and thereby promoting selective translation of mRNAs to restore homeostasis. Despite the importance of the ISR and intensive study over the past decades, structural insight into how Gcn2 interacts with ribosomal particles has been lacking. Using ex vivo affinity purification approaches, we have obtained a native structure of a Gcn2 dimer in complex with the ribosomal 60S subunit. The Gcn2 dimer is formed by dimerization of the histidine tRNA synthetase-like domains, which establish extensive interactions with the stalk-base and sarcin-ricin loop of the 60S subunit. The C-terminal domain of Gcn2 is also dimerized and occupies the A- and P-site tRNA binding sites at the peptidyl-transferase center of the 60S subunit. Complementary functional studies indicate that binding of Gcn2 to the 60S subunit does not require the co-activators Gcn1 or Gcn20, nor does it lead to phosphorylation of eIF2a. Rather, upon stress, we observe a shift of Gcn2 from the 60S subunit into the colliding ribosome fraction, suggesting that the Gcn2-60S complex represents an inactive stand-by state to enable a rapid redistribution to collided ribosomes, and thereby facilitating a quick and efficient response to stress.
2024-09-16 | PXD055898 | Pride
Project description:Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder, caused by an unstable CAG-repeat expansion in the SCA2 gene, which encodes a polyglutamine (polyQ) domain expansion in the protein ataxin-2 (ATXN2). The RNA-binding protein ATXN2 interacts with the poly(A)-binding protein PABPC1, localizing to ribosomes in the rough endoplasmic reticulum or in polysomes. Under cell stress ATXN2 and PABPC1 are relocated to stress granules where mRNAs are protected from translation and from degradation. It is unknown whether ATXN2 associates preferentially with specific mRNAs or how it modulates their processing. Here, we investigated the RNA profile of liver and cerebellum from adult Atxn2 knock-out (Atxn2-/-) mice, employing oligonucleotide microarrays for screening and RNA deep sequencing for validation. We consistently observed modest upregulations for the level of many mRNAs encoding proteins of the ribosomal large and small subunit as well as the translation initiation complex. Quantitative reverse transcriptase PCR in liver tissue confirmed these upregulations for the ribosomal components Rpl14, Rpl18, Rps10, Rps18, Gnb2l1, the translation initiation factors Eif2s2, Eif3s6, Pabpc1, and the ribosomal biogenesis modulator Nop10. Quantitative immunoblots substantiated the effects for PABPC1. Thus, the physiological role of ATXN2 modifies the abundance of cellular translation factors.
2017-05-27 | GSE57998 | GEO
Project description:The raw proteomics data in the manuscript "Quantitative proteomics reveals metabolic reprogramming in host cells induced by trophozoites and intermediate subunit of Gal/GalNAc lectins from Entamoeba histolytica".
2022-03-01 | PXD031959 | JPOST Repository
Project description:Hcr1/eIF3j is a sub-stoichiometric subunit of eukaryotic initiation factor 3 (eIF3) that can dissociate the post-termination 40S ribosomal subunit from mRNA in vitro. We examined this ribosome recycling role in vivo by ribosome profiling and reporter assays and found that loss of Hcr1 led to reinitiation of translation in 3’UTRs, consistent with a defect in recycling. However, the defect appeared to be in recycling of the 60S subunit, rather than the 40S subunit, because reinitiation did not require an AUG codon and was suppressed by overexpression of the 60S dissociation factor Rli1/ABCE1. Consistent with a 60S recycling role, overexpression of Hcr1 could not compensate for loss of 40S recycling factors Tma64/eIF2D and Tma20/MCT-1. Intriguingly, loss of Hcr1 triggered higher expression of RLI1 via an apparent feedback loop. These findings suggest Hcr1/eIF3j is recruited to ribosomes at stop codons and may coordinate the transition to a new round of translation.
2019-05-23 | GSE124204 | GEO