Genome wide mapping of Npa1-HTP binding sites in yeast by CRAC
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PROVIDER: PRJNA412225 | ENA |
REPOSITORIES: ENA
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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: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 Npa1, ribosome assembly factors, on RNAs in vivo.
2018-08-07 | GSE104281 | GEO
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: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:Purpose: The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. We applied an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between individual yeast exosome subunits and their targets in a living cell. Methods: We apply CRAC to HTP-tagged proteins (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A): Two nucleases (Rrp44, Rrp6) and two structural subunits (Rrp41, Csl4) of the yeast exosome. At least two independent experiments were performed in each case and analyzed separately. We performed CRAC on wild-type (WT) Rrp44 and two catalytic mutants, rrp44-endo (D91N, E120Q, D171N, D198N) and rrp44-exo (D551N). We further developed CRAC using cleavable proteins (split-CRAC) to compare endonuclease and exonuclease targets of Rrp44. Plasmids designed for split-CRAC contain a PreScission protease cleavage site (PP) inserted between aa 241 and 242 in the RRP44 ORF to allow in vitro cleavage of purified protein, and a His6 tag to select the respective cleaved fragment. Results: Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of noncoding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome. This indicates that substrates can follow multiple pathways to the nucleases. Conclusion: Our study represents the first transcriptome-wide map of substrates for the yeast exosome nuclease complex. Identification of targets for individual exosome subunits in wild-type and mutant yeast cells.
2012-09-25 | E-GEOD-40046 | biostudies-arrayexpress
Project description:Purpose: The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. We applied an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between individual yeast exosome subunits and their targets in a living cell. Methods: We apply CRAC to HTP-tagged proteins (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A): Two nucleases (Rrp44, Rrp6) and two structural subunits (Rrp41, Csl4) of the yeast exosome. At least two independent experiments were performed in each case and analyzed separately. We performed CRAC on wild-type (WT) Rrp44 and two catalytic mutants, rrp44-endo (D91N, E120Q, D171N, D198N) and rrp44-exo (D551N). We further developed CRAC using cleavable proteins (split-CRAC) to compare endonuclease and exonuclease targets of Rrp44. Plasmids designed for split-CRAC contain a PreScission protease cleavage site (PP) inserted between aa 241 and 242 in the RRP44 ORF to allow in vitro cleavage of purified protein, and a His6 tag to select the respective cleaved fragment. Results: Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of noncoding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome. This indicates that substrates can follow multiple pathways to the nucleases. Conclusion: Our study represents the first transcriptome-wide map of substrates for the yeast exosome nuclease complex.
2012-09-25 | GSE40046 | GEO
Project description:We quantified the exact RNA binding sites of the Ssd1 protein in Saccharomyces cerevisiae, in exponential growth and heat shock conditions, using the CRAC protocol. We used a His-TEV-protein A tag (HTP) on the C-terminal of the genomic copy of Ssd1, with the 3'UTR replaced by the 3'UTR/terminator from the K. lactis Ssd1 homolog, followed by a KlURA3 selection marker.
2020-10-23 | GSE159835 | GEO
Project description:CRAC of Nab3 in yeast cells accumulating a circular RNA containing Nab3 binding sites (the decoy) or an empty control
| PRJNA691262 | ENA