High-throughput measurements of fitness effects of mutations in the yeast U3 snoRNA
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ABSTRACT:
ORGANISM(S): synthetic construct Saccharomyces cerevisiae
PROVIDER: GSE77709 | GEO | 2016/02/09
SECONDARY ACCESSION(S): PRJNA311244
REPOSITORIES: GEO
Similar Datasets
Project description:We used a molecular barcoding approach to quantify the fitness of ~60,000 randomly mutated variants of the Saccharomyces cerevisiae U3 snoRNA (the data were further used to calculate a map of intragenic epistatic interactions within this RNA molecule, as descibed in Puchta et al. 2016) We generated two independent mutant libraries ('Small' with ~3 and 'Big' with ~10 SNPs per allele) on centromeric plasmid, with 20nt random barcodes placed in a non-transcribed region downstream of the gene (PE Samples 30-37 - paired-end sequencing of: U3, 70nt linker and 20nt barcode); next we performed competiton experiments in several conditions (E1-5) on populations of yeast cells transformed with both libraries and measured frequencies of mutated variants in time-points during the experiment by deep sequencing of barcodes (Samples 1-29).
2016-02-09 | E-GEOD-77709 | biostudies-arrayexpress
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:Small nucleolar RNAs (snoRNAs) are prevailing components of the chromatin-associated transcriptome. We have studied a specific chromatin-associated snoRNA, snoRNA:U3:9B, that binds to a large number of protein coding genes in the genome of Drosophila melanogaster, including immune response genes. We have used CRISPR/Cas9 to delete snoRNA:U3:9B. SnoRNA:U3:9B-deficient larvae were viable in control conditions but failed to develop into pupae when challenged by expression of a Sindbis virus replicon, which suggests that snoRNA:U3:9B is required for the activation of an effective antiviral response. Consistent with this hypothesis, the chromatin decompaction and gene activation typically observed at immune response genes in response to Sindbis replicon expression were abolished in snoRNA:U3:9B-deficient larvae. Moreover, snoRNA:U3:9B was required for the recruitment of the ATP-dependent helicase brm to a set of target immune genes. The association of snoRNA:U3:9B with immune response genes in vivo shown by ChIRP-qPCR suggests that the chromatin and gene expression defects observed in the snoRNA:U3:9B knock-out strains are due to direct regulatory events. In summary, our findings reveal an immune defence mechanism that relies on a snoRNA for the recruitment of chromatin remodelling factors to immune genes, thereby modulating local chromatin accessibility to facilitate immune gene activation.
2025-07-29 | GSE281409 | GEO
Project description:Small nucleolar RNAs (snoRNAs) are prevailing components of the chromatin-associated transcriptome. We have studied a specific chromatin-associated snoRNA, snoRNA:U3:9B, that binds to a large number of protein coding genes in the genome of Drosophila melanogaster, including immune response genes. We have used CRISPR/Cas9 to delete snoRNA:U3:9B. SnoRNA:U3:9B-deficient larvae were viable in control conditions but failed to develop into pupae when challenged by expression of a Sindbis virus replicon, which suggests that snoRNA:U3:9B is required for the activation of an effective antiviral response. Consistent with this hypothesis, the chromatin decompaction and gene activation typically observed at immune response genes in response to Sindbis replicon expression were abolished in snoRNA:U3:9B-deficient larvae. Moreover, snoRNA:U3:9B was required for the recruitment of the ATP-dependent helicase brm to a set of target immune genes. The association of snoRNA:U3:9B with immune response genes in vivo shown by ChIRP-qPCR suggests that the chromatin and gene expression defects observed in the snoRNA:U3:9B knock-out strains are due to direct regulatory events. In summary, our findings reveal an immune defence mechanism that relies on a snoRNA for the recruitment of chromatin remodelling factors to immune genes, thereby modulating local chromatin accessibility to facilitate immune gene activation.
2025-07-29 | GSE281900 | GEO
Project description:Comparison of endogenous gene expression differences between control and overexpressing U3 snoRNA in HPF.
2022-01-08 | GSE193086 | 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: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: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:High-throughput measurements of fitness effects of mutations in the yeast U3 snoRNA
| PRJNA311244 | ENA
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