RNA binding of Saccharomyces cerevisiae Ssd1
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae BY4741
PROVIDER: GSE159835 | GEO | 2020/10/23
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: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: 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: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:The speB gene of Streptococcus pyogenes has a long 5' UTR with unknown functions. Preliminary studies showed that the partial mutation of this region led to altered expression patterns of the speB gene and ropB, its upstream gene. In this study, we used microarray to examine the effect of speB 5' UTR mutation on the S. pyogenes transcriptome. The speB ORF and a large portion of the speB 5’ UTR (-1 to -527 from ATG) was replaced with an erythromycin resistant cassette gene (ermAM) to generate a ΔIG mutant. Transcriptome profiles of the wild-type (WT) and ΔIG mutant in the mid-exponential phase and early stationary phase (ST) were examined with microarray analysis.
2015-09-10 | E-GEOD-72838 | biostudies-arrayexpress
Project description:The response of L. lactis to the presence of S. cerevisiae was analyzed during the exponential growth phase in fermentors in defined growth conditions. Although no growth kinetic difference was observed between the pure and mixed culture of L. lactis, the mRNA level of genes was significantly modified. More particularly, a strong reorientation of pyrimidine metabolism was observed when L. lactis was grown in the mixed culture. Keywords: microbial interaction, time course
2008-10-23 | GSE9436 | GEO
Project description:SSD1 is a polymorphic locus in budding yeast with many pleiotropic effects. Our lab had previously done transcript microarray of W303a in an ssd1-d background, and here we have carried out another transcript microarray across the cell cycle in an isogenic SSD1-V background. We find that a large fraction of budding yeast transcripts is differentially expressed in these cells. Ssd1 has recently been shown to bind mRNAs in vivo, but very few of these mRNAs show significant changes in levels in the SSD1-V versus ssd1-d comparison. About 20% of cell cycle-regulated transcripts are affected by SSD1-V and most of these transcripts show sharper amplitudes of oscillation in SSD1-V cells. Many transcripts whose gene products influence longevity are also affected, the largest class of which are involved in translation. Ribosomal protein mRNAs are globally down-regulated by SSD1-V. Cells were synchronized with alpha factor and sampled every 10 min across 2 cell cycles. A total of 13 samples were analyzed. No replicates were included. cDNA of the cell cycle samples were labelled with Cy5. For Cy3 labelling, asynchronous yeast population was used.
2010-05-06 | E-GEOD-16911 | biostudies-arrayexpress
Project description:The global transcriptome of the Bifidobacterium animalis subsp. lactis Bl-04 was analyzed during exponential growth on 11 prebiotic carbohydrates and glucose to identify the specific gene cluster differentially upregulated in response to each carbohydrate.
2013-10-23 | GSE41906 | GEO
Project description:SSD1 is a polymorphic locus in budding yeast with many pleiotropic effects. Our lab had previously done transcript microarray of W303a in an ssd1-d background, and here we have carried out another transcript microarray across the cell cycle in an isogenic SSD1-V background. We find that a large fraction of budding yeast transcripts is differentially expressed in these cells. Ssd1 has recently been shown to bind mRNAs in vivo, but very few of these mRNAs show significant changes in levels in the SSD1-V versus ssd1-d comparison. About 20% of cell cycle-regulated transcripts are affected by SSD1-V and most of these transcripts show sharper amplitudes of oscillation in SSD1-V cells. Many transcripts whose gene products influence longevity are also affected, the largest class of which are involved in translation. Ribosomal protein mRNAs are globally down-regulated by SSD1-V.
2009-09-01 | GSE16911 | GEO