UtpA and UtpB chaperone nascent pre-ribosomal RNA and the U3 snoRNP to initiate eukaryotic ribosome assembly
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE79950 | GEO | 2016/09/21
SECONDARY ACCESSION(S): PRJNA317481
REPOSITORIES: GEO
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Project description:The SSU Processome (sometimes referred to as 90S) is an early stable intermediate in the small ribosomal subunit biogenesis pathway of eukaryotes. Progression of the SSU Processome to a pre-40S particle requires a large-scale compaction of the RNA and release of many biogenesis factors. The U3 snoRNA is a primary component of the SSU Processome and hybridizes to the rRNA at multiple locations to organize the structure of the SSU Processome. Thus, release of U3 is prerequisite for the transition to pre-40S. Our lab proposed that the RNA helicase Dhr1 plays a crucial role in the transition by unwinding U3 and that this activity is controlled by the SSU Processome protein Utp14. How Utp14 times the activation of Dhr1 is an open question. Despite being highly conserved, Utp14 contains no recognizable domains, and how Utp14 interacts with the SSU Processome is not well characterized. Here, we used UV crosslinking and analysis of cDNA (CRAC) and yeast two-hybrid interaction to characterize how Utp14 interacts with the pre-ribosome. Moreover, proteomic analysis of SSU particles lacking Utp14 revealed that the presence of Utp14 is needed for efficient recruitment of the RNA exosome. Our analysis positions Utp14 to be uniquely poised to communicate the status of assembly of the SSU Processome to Dhr1 and possibly to the exosome as well.
2018-06-21 | GSE115748 | GEO
Project description:SIRT7 is an NAD+-dependent protein deacetylase with important roles in ribosome biogenesis and cell proliferation. Previous studies have established that SIRT7 is associated with RNA polymerase I, interacts with pre-rRNA and promotes rRNA synthesis. Here we show that SIRT7 is also associated with snoRNAs that are involved in pre-rRNA processing and rRNA maturation. Knockdown of SIRT7 impairs U3 snoRNA-dependent early cleavage steps that are necessary for generation of 18S rRNA. Mechanistically, SIRT7 deacetylates U3-55k, a core component of the U3 snoRNP complex, and reversible acetylation of U3-55k modulates the association of U3-55k with U3 snoRNA. Deacetylation by SIRT7 enhances U3-55k binding to U3 snoRNA, which is a prerequisite for pre-rRNA processing. Under stress conditions, SIRT7 is released from nucleoli, leading to hyperacetylation of U3-55k and attenuation of prerRNA processing. The results reveal a multifaceted role of SIRT7 in ribosome biogenesis, regulating both transcription and processing of rRNA. CLIP-seq was performed in Flag-SIRT7-293T cells.
2016-01-07 | E-GEOD-76578 | biostudies-arrayexpress
Project description:SIRT7 is an NAD+-dependent protein deacetylase with important roles in ribosome biogenesis and cell proliferation. Previous studies have established that SIRT7 is associated with RNA polymerase I, interacts with pre-rRNA and promotes rRNA synthesis. Here we show that SIRT7 is also associated with snoRNAs that are involved in pre-rRNA processing and rRNA maturation. Knockdown of SIRT7 impairs U3 snoRNA-dependent early cleavage steps that are necessary for generation of 18S rRNA. Mechanistically, SIRT7 deacetylates U3-55k, a core component of the U3 snoRNP complex, and reversible acetylation of U3-55k modulates the association of U3-55k with U3 snoRNA. Deacetylation by SIRT7 enhances U3-55k binding to U3 snoRNA, which is a prerequisite for pre-rRNA processing. Under stress conditions, SIRT7 is released from nucleoli, leading to hyperacetylation of U3-55k and attenuation of prerRNA processing. The results reveal a multifaceted role of SIRT7 in ribosome biogenesis, regulating both transcription and processing of rRNA.
2016-01-07 | GSE76578 | GEO
Project description:Ribosome biosynthesis plays a crucial role in regulating protein translation and is essential for cell growth and development in physiological progress. The progression and recurrence of Pterygia mainly occur due to the abnormal proliferation and migration of stromal Pterygia fibroblasts. Small nucleolar RNA U3 (U3 snoRNA), harboring the atypical C/D boxes, is involved in 18S ribosomal RNA (18S rRNA) synthesis; however, the mechanism of U3 snoRNA in Pterygia remains unclear. Methods: Primary HCFs and HPFs were separated and cultured from fresh conjunctival grafts and Pterygia tissues. The PLKO.1 lentiviral system and CRISPR/Cas9 recombinant construct were, respectively, used to overexpress and silence U3 snoRNA in HPF and HCF cells for further specific phenotypes analysis. RNA-seq and TMT-labeled quantitative protein mass spectrometry were utilized to evaluate the effect of U3 snoRNA on mRNA transcripts and protein synthesis. Results: Reduced U3 snoRNA in Pterygia promotes HCF or HPF cells' proliferation, migration, and cell cycle but has no significant effect on apoptosis. U3 snoRNA modulates 18S rRNA synthesis through shearing precursor ribosomal RNA 47S rRNA at the 5′ external transcribed spacer (5′ ETS). Moreover, the altered U3 snoRNA causes mRNA and protein differential expression in HCF or HPF cells. Conclusion: The atypical U3 snoRNA regulates the translation of specific proteins to exert a suppressive function in Pterygia through modulating the 18S RNA synthesis. Here, we uncover a novel insight into U3 snoRNA biology in the development of Pterygia.
2022-05-20 | PXD030801 | Pride
Project description:In eukaryotes, biogenesis of ribosomes requires folding and assembly of the precursor rRNA (pre-rRNA) with a large number of proteins and snoRNPs into huge RNA-protein complexes. In spite of intense genetic, biochemical and high resolution cryo-EM studies in Saccharomyces cerevisiae, information about the conformation of the earliest 35S pre-rRNA is limited. To overcome this, we performed high-throughput SHAPE chemical probing on the 35S pre-rRNA associated with 90S pre-ribosomes. We focused our analyses on external (5´ETS) and internal (ITS1) transcribed spacers as well as the 18S region. We show that in the 35S pre-rRNA, the central region of the 18S is in a more open configuration compared to 20S pre-rRNA and that the central pseudoknot is not formed. The essential ribosome biogenesis protein Mrd1 influences the structure of the 18S part locally and is involved in organizing the central pseudoknot and surrounding structures. Our results demonstrate that the U3 snoRNA dynamically interacts with the 35S pre-rRNA and that Mrd1 is required for disrupting U3 snoRNA base-pairing interactions in the 5'ETS. We propose that the dynamic U3 snoRNA interactions and Mrd1 are essential for establishing the structure of the central region of 18S that is required for processing and 40S subunit function.
2018-01-15 | GSE106868 | GEO
Project description:Osteoarthritis (OA) is a chronic debilitating joint disease which is strongly associated with ageing. OA involves pathological cellular processes in all joint structures and affects articular cartilage integrity, leading to dysfunctional joint articulation. The biomolecular processes that catalyze the disturbances in the articular chondrocyte phenotype leading to OA are poorly understood, and it is expected that a comprehensive understanding of the avenues leading to catabolic changes and disruption of articular chondrocyte homeostasis will provide important cues for future treatments of the condition. Chondrocytes are specialized secretory cells with highly active protein translational machinery, enabling the synthesis and maintenance of the protein-rich cartilage extracellular matrix (ECM). Disturbances in chondrocyte protein translation in cartilage development and OA are connected to mTOR activity, ER stress, unfolded protein response (UPR)and CHOP-mediated apoptosis. These responses change the downstream translational activity of the biosynthesized ribosome. The assembled mammalian ribosome is built from ribosomal RNAs (rRNAs), together with more than 80 different protein subunits. At the heart of the ribosome, the 18S rRNA guides the decoding of the mRNA message, while an ancient ribozyme activity in the 28S rRNA forms the core of the peptidyltransferase center that polymerizes the amino acid sequence encoded by the mRNA into functional proteins. Post-transcriptional maturation of rRNAs is an integral part of the biosynthesis of ribosomes and ribonucleolytic processing of the major 47S rRNA precursor into mature 18S, 5.8S, and 28S rRNAs is rate limiting for ribosome biogenesis. The U3 small nucleolar RNA (snoRNA) is an evolutionarily highly conserved box C/D-class snoRNA which catalyzes the endoribonucleolytic processing of the 5’ external transcribed spacer (ETS) of the 47S pre-rRNA by base complementarity-guided pre-rRNA substrate recognition and plays a crucial role in the maturation of 18S rRNA. Although extensively studied in yeast, it was only recently demonstrated that U3 snoRNA is indispensable for rRNA maturation in human cells. Pathways controlling ribosome activity have previously been described in the regulation of chondrocyte homeostasis. We here now postulate that not only ribosome activity is involved in chondrocyte homeostasis, but that OA pathophysiological situations can also cause alterations in chondrocyte ribosome biogenesis with consequences for cellular protein translation. Since U3 snoRNA-driven rRNA production is rate-limiting in ribosome biogenesis, we hypothesized that the U3 snoRNA is critical for chondrocyte homeostasis. In this study we therefor aimed to determine whether OA pathophysiological conditions interact with chondrocyte U3 snoRNA levels, thereby influencing rRNA levels and chondrocyte translation capacity.
2020-08-21 | PXD017253 | Pride
Project description:Eukaryotic ribosome biogenesis begins with the co-transcriptional assembly of the 90S pre-ribosome. The ‘U three protein’ (UTP) complexes and snoRNA particles arrange around the nascent pre-ribosomal RNA chaperoning its folding and further maturation. The earliest event in this hierarchical process is the binding of the UTP-A complex to the 5’-end of the pre-ribosomal RNA (5’-ETS). This oligomeric complex predominantly consists of β-propeller and α-solenoidal proteins. Here we present the structure of the Utp4 subunit from the thermophilic fungus Chaetomium thermophilum at 2.15 Å resolution and analyze its function by UV RNA-crosslinking (CRAC) and in context of a recent cryo-EM structure of the 90S pre-ribosome. Utp4 consists of two orthogonal and highly basic β-propellers that perfectly fit the EM-data. The Utp4 structure highlights an unusual Velcro-closure of its C-terminal β-propeller as relevant for protein integrity and Utp8 recognition in the context of the pre-ribosome. We provide a first model of the 5’-ETS RNA from an internally hidden 5’-end up to the region that hybridizes to the 3’-hinge sequence of U3 snoRNA and validate a specific Utp4/5’-ETS interaction by CRAC analysis. Altogether Utp4 is central to the UTP-A complex and organizes the 5’-ETS for further maturation.
2017-06-06 | GSE98331 | GEO
Project description:UtpA and UtpB chaperone nascent pre-ribosomal RNA and the U3 snoRNP to initiate eukaryotic ribosome assembly
| PRJNA317481 | ENA
Project description:Ribosome biogenesis, which takes place mainly in the nucleolus, involves coordinated expression of pre-ribosomal RNAs (pre-rRNAs) and ribosomal proteins, pre-rRNA processing, and subunit assembly with the aid of numerous assembly factors. Our previous study showed that the Arabidopsis thaliana protein arginine methyltransferase AtPRMT3 regulates pre-rRNA processing; however, the underlying molecular mechanism remains unknown. Here, we report that AtPRMT3 interacts with Ribosomal Protein S2 (RPS2), facilitating processing of the 90S/Small Subunit (SSU) processome and repressing nucleolar stress. We isolated an intragenic suppressor of atprmt3-2, which rescues the developmental defects of atprmt3-2 while produces a putative truncated AtPRMT3 protein bearing the entire N-terminus but lacking an intact enzymatic activity domain. We further identified RPS2 as an interacting partner of AtPRMT3, and found that loss-of-function rps2a2b mutants were phenotypically reminiscent of atprmt3, showing pleiotropic developmental defects and aberrant pre-rRNA processing. RPS2B binds directly to pre-rRNAs in the nucleus, and such binding is enhanced in atprmt3-2. Consistently, multiple components of the 90S/SSU processome were more enriched by RPS2B in atprmt3-2, which accounts for early pre-rRNA processing defects and results in nucleolar stress. Collectively, our study uncovered a novel mechanism by which AtPRMT3 cooperates with RPS2B to facilitate the dynamic assembly/disassembly of the 90S/SSU processome during ribosome biogenesis and repress nucleolar stress.
2020-12-03 | GSE140318 | GEO
Project description:Sulfolobus solfataricus 98/2 U3 transcriptome - U3 RNA
| PRJNA424779 | ENA