Illumina sequencing of CRAC data obtained with Ylr419w
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE252043 | GEO | 2024/01/08
REPOSITORIES: GEO
Similar Datasets
Project description:Purpose: Pre-ribosomal RNA is cleaved at defined sites to release the mature ribosomal RNAs, but the functions of many ribosome biogenesis factors involved in 18S rRNA release are not known. We apply an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between the yeast PIN domain protein Utp23 and its targets in a living cell. Methods: We apply CRAC to an HTP-tagged Utp23 protein (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A) in budding yeast. At least two independent experiments were performed and analysed separately. A non-tagged yeast strain was also used as a negative control. Results: We found that yeast Utp23 UV-crosslinked in vivo to the snR30 snoRNA and to the eukaryotic-specific expansion segment 6 (ES6) in the 18S rRNA. Conclusion: According to our crosslinking data, Utp23 is perfectly positioned to coordinate release of the snR30 snoRNA from the 18S ES6 region.
2017-01-11 | GSE87238 | GEO
Project description:Box C/D snoRNAs snR4 and snR45, whose role was unknown, guide 18S rRNA acetylations by Kre33 in yeast. UV-crosslinking (CRAC) was used to determine binding sites of Kre33 on RNA.
2017-07-25 | GSE87480 | GEO
Project description:We report the application of the Cross-Linking and cDNA (CRAC) technique to identify binding sites of Npa1, ribosome assembly factors, on RNAs in vivo.
2018-08-07 | GSE104281 | GEO
Project description:We report the application of the Cross-Linking and cDNA (CRAC) technique to identify binding sites of RBP95, ribosome assembly factors, on RNAs in vivo. Once sequenced, Rnas associated have been aligned to the yeast genomes and enrichment of specific RNAs has been studied in more details. Specifically apparition of Mutation/deletion in the reads are of interesdt since they mostly corresponds to prceise site of cross-link between bait protein and target RNA. Here RBP95-HTP is mostly bound to 25S rRNA at one partiuclar site and to several snoRNAs, including SNR37, SNR30, SNR190, SNR86 and SNR128.
2022-08-23 | GSE189589 | GEO
Project description:We report the application of the Cross-Linking and cDNA (CRAC) technique to identify binding sites of DBP6, ribosome assembly factors, on RNAs in vivo. Once sequenced, RNAs associated have been aligned to the yeast genomes and enrichment of specific RNAs has been studied in more details. Specifically apparition of Mutation/deletion in the reads are of interesdt since they mostly corresponds to prceise site of cross-link between bait protein and target RNA. Here DBP6-HTP is mostly bound to snoRNAs (SNR30, SNR10, SNR42, SRN3,SRN190 and SNR13) and 25S rRNA around the same binding sites of the identifed snoRNAs.
2022-12-05 | GSE200692 | GEO
Project description:Purpose: Pre-ribosomal RNA is cleaved at defined sites, but many endonucleases involved in 18S rRNA release are not known. We apply an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between a yeast candidate pre-rRNA endonuclease (Utp24) and its targets in a living cell. Methods: We apply CRAC to an HTP-tagged Utp24 protein (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A). At least two independent experiments were performed and analyzed separately. Results: We found that yeast Utp24 UV-crosslinked in vivo to the U3 snoRNA and all (pre-)rRNA elements that form the central pseudoknot in the 18S rRNA. The pseudoknot is an evolutionarily highly conserved structure that is required to ensure pre-rRNA processing at three cleavage sites (A0, A1 and A2) and still present in the mature rRNA. According to our crosslinking data, the endonuclease Utp24 is placed in close proximity to site A1 at the 5'-end of the 18S rRNA. Conclusion: Our study strongly supports the hypothesis that Utp24 cleaves pre-rRNA at sites A1 and A2. Examination of targets for pre-rRNA endonucleases in yeast cells.
2016-04-01 | E-GEOD-75991 | biostudies-arrayexpress
Project description:Purpose: Pre-ribosomal RNA is cleaved at defined sites, but many endonucleases involved in 18S rRNA release are not known. We apply an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between a yeast candidate pre-rRNA endonuclease (Utp24) and its targets in a living cell. Methods: We apply CRAC to an HTP-tagged Utp24 protein (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A). At least two independent experiments were performed and analyzed separately. Results: We found that yeast Utp24 UV-crosslinked in vivo to the U3 snoRNA and all (pre-)rRNA elements that form the central pseudoknot in the 18S rRNA. The pseudoknot is an evolutionarily highly conserved structure that is required to ensure pre-rRNA processing at three cleavage sites (A0, A1 and A2) and still present in the mature rRNA. According to our crosslinking data, the endonuclease Utp24 is placed in close proximity to site A1 at the 5'-end of the 18S rRNA. Conclusion: Our study strongly supports the hypothesis that Utp24 cleaves pre-rRNA at sites A1 and A2.
2016-04-01 | GSE75991 | GEO
Project description:Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4+ T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (Mixed Lymphocyte Reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC inhibitor that potently inhibits human T cell functions. PBMC from healthy donors (n=4) were stimulated with anti-CD3/CD28 in the presence or absence of CRAC inhibitor, CsA or FK506 for 24 hrs
2013-01-31 | E-GEOD-43210 | biostudies-arrayexpress
Project description:Systemic lupus erythematous (SLE) is a prototype of autoimmune disease. Lupus nephritis (LN) is one of the most serious complications of SLE. The development of autoreactive B cells and the production of autoantibodies have been critical for the initiation and progression of LN. Store-operated Ca2+ entry (SOCE) is the main Ca2+ influx pathway in lymphocytes and is essential for immune response . SOCE is mediated by Ca2+ release-activated Ca2+ (CRAC) channels which are comprised of stromal interaction molecule (STIM) and calcium release-activated calcium modulators (ORAI) . Mutations in genes encoding the CRAC channel abolish SOCE in cells of the immune system and cause severe combined immunodeficiency . Calcium signaling via ORAI1 has been involved in the pathogenesis of autoimmune diseases by driving Th17 differentiation . STIM1 deficiency significantly reduced Th1/Th17 responses and resulted in complete protection from experimental autoimmune encephalomyelitis . Compared to T cells, the roles of CRAC channel in B cells is far less clear. Ca²⁺/calmodulin (CaM)-dependent protein kinase2 (CaMK2) is a serine/threonine-specific protein kinase that is regulated by the Ca²⁺/CaM complex. CaMK2 has been involved in many signaling pathways and is necessary for Ca²⁺ homeostasis, T cell development and activation. CaMK4, another member of the CaMK family, has been shown to compromises podocyte function and promote renal diseases in LN. In the current study, we found that CRAC channel mediated calcium signaling is enhanced in B cells from patients with LN. CRAC channel inhibition by YM-58483 and knocked down of CRAC channel by ORAI1 or STIM2 siRNA led to suppression of CaMK2 signaling and decreased B cell differentiation. Lupus mice treated with CRAC channel inhibitor showed reduced anti-double stranded DNA antibodies (anti-dsDNA), decreased immune deposition in the glomeruli and improved renal function. CRAC channel mediates the development and progression of LN by promoting the differentiation of B cells into plasma cells.
2021-09-01 | GSE175424 | GEO
Project description:Ribosome biogenesis is essential for protein synthesis in gene expression. Yeast eIF5B has been shown biochemically to facilitate 18S rRNA 3’ end maturation during late-40S ribosomal subunit assembly and gate the transition from translation initiation to elongation. But the effects of eIF5B have not been studied at the genome-wide level in any organism, and 18S rRNA 3’ end maturation is poorly understood in plants. Arabidopsis HOT3/eIF5B1 was found to promote development and heat-stress acclimation by translational regulation, but its molecular function remained unknown. Here, we show that HOT3 is a late-stage ribosome biogenesis factor that facilitates 18S rRNA 3’ end processing and is a translation initiation factor that globally impacts the transition from initiation to elongation. By developing and implementing 18S-ENDseq, we revealed previously unknown events in 18S rRNA 3’ end maturation or metabolism. We quantitatively defined new processing hotspots and identified adenylation as the prevalent non-templated RNA modification at the 3’ ends of pre-18S rRNAs. Aberrant 18S rRNA maturation in hot3 further activated RNAi to generate RDR1- and DCL2/4-dependent risiRNAs mainly from a 3’ portion of 18S rRNA. We further showed that risiRNAs in hot3 were predominantly localized in ribosome-free fractions not responsible for the 18S rRNA maturation or translation initiation defects in hot3. Our study uncovered the molecular function of HOT3/eIF5B1 in 18S rRNA maturation at the late-40S assembly stage and revealed the regulatory crosstalk among ribosome biogenesis, mRNA translation initiation, and siRNA biogenesis in plants.
2023-04-19 | GSE223168 | GEO