Polyadenylation site mapping of xrn1∆ in Sacchromyces cerevisiae
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PROVIDER: PRJNA665542 | ENA |
REPOSITORIES: ENA
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Project description:The use of alternative polyadenylation sites is common and affects the post-transcriptional fate of mRNA, including its stability, localization, and translation. Here we use the internal version of our previously developed protocol (PMID: 23295673), to characterize the polyA sites in a xrn1∆ strain.
2020-09-25 | GSE158548 | GEO
Project description:The use of alternative polyadenylation sites is common and affects the post-transcriptional fate of mRNA, including its stability, localization, and translation. Here we present a method for genome-wide and strand-specific mapping of poly(A) sites and quantification of RNA levels at unprecedented efficiency by using an on-cluster dark T-fill procedure on the Illumina sequencing platform. Our method outperforms former protocols in quality and throughput, and reveals new insights into polyadenylation in Saccharomyces cerevisiae. Experimental benchmark of five different protocols (3tfill, bpmI, internal, rnaseq and yoon) for genome-wide identification of polyadenylation sites in Saccharomyces cerevisiae and transcript quantification. RNA was extracted from WT cells grown in glucose (ypd) or galactose (ypgal) as carbon source. The same RNA was used for 3 independent library constructions (technical replicates, rep).
2013-01-10 | E-GEOD-40110 | biostudies-arrayexpress
Project description:The use of alternative polyadenylation sites is common and affects the post-transcriptional fate of mRNA, including its stability, localization, and translation. Here we present a method for genome-wide and strand-specific mapping of poly(A) sites and quantification of RNA levels at unprecedented efficiency by using an on-cluster dark T-fill procedure on the Illumina sequencing platform. Our method outperforms former protocols in quality and throughput, and reveals new insights into polyadenylation in Saccharomyces cerevisiae.
2013-01-10 | GSE40110 | GEO
Project description:mRNA homeostasis is favored by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5’ exhibited significant reductions in slope that were compatible with decreased elongation rates. This phenomenon was also observed soon after the nuclear pool of Xrn1 was depleted. Nucleosome mapping confirmed drastically altered chromatin dynamics. We also found an accumulation of arrested RNA pol II at the 3’ end, immediately upstream of polyadenylation sites of most genes, with particular incidence in those functionally related to regulatory processes. Lack of Xrn1 provoked alterations in RNA pol II CTD phosphorylation and increased recruitment of 3’ pre-mRNA processing factors. However, accumulation of RNA pol II at 3’ ends was rather related to the nucleosomal configuration of those regions.
2021-05-24 | GSE153036 | GEO
Project description:mRNA homeostasis is favored by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5’ exhibited significant reductions in slope that were compatible with decreased elongation rates. This phenomenon was also observed soon after the nuclear pool of Xrn1 was depleted. Nucleosome mapping confirmed drastically altered chromatin dynamics. We also found an accumulation of arrested RNA pol II at the 3’ end, immediately upstream of polyadenylation sites of most genes, with particular incidence in those functionally related to regulatory processes. Lack of Xrn1 provoked alterations in RNA pol II CTD phosphorylation and increased recruitment of 3’ pre-mRNA processing factors. However, accumulation of RNA pol II at 3’ ends was rather related to the nucleosomal configuration of those regions.
2021-05-24 | GSE153035 | GEO
Project description:Purpose: To determine genes that undergo alternative polyadenylation in proliferating versus quiescent fibroblasts. Methods: Three different biological replicates (fibroblast strains, 10-1, 12-1, and 12-2) were used for generating proliferating and quiescent (7-day contact inhibited and 7-day serum-starved) cells. RNA extracted from these cells were used for library preparation. cDNA fragments were enriched for the junction between the poly(A) site and the end of the 3' UTR using a modified high throughput sequencing protocol (GnomeGen). cDNA libraries were created according to Gnome-Gen RNA-seq library preparation kit for RNA profiling except Amgen Ampure XP beads were used instead of the Gnome-gen size selector product to remove ligation reaction products before proceeding to the reverse transcription step. The libraries were sequenced on an Illumina HiSeq 2000 instrument. The sequencing reaction wasa run for 147 cycles. Reads from pol(A) enriched cDNA libraries were aligned to the genome using the STAR alignment algorithm after the computational removal of untemplated adenosines. Results: Polyadenylation site selection was significantly altered in approximately 10% of genes in quiescent compared with proliferating fibroblasts. Contact inhibited and serum starved fibroblasts had similar polyadenylation site selection profiles. Conclusions: Quiescence is associated with changes in polyadenylation site selection.
2018-07-19 | GSE117337 | GEO
Project description:Antisense (as)lncRNAs are extensively degraded by the nuclear exosome and the cytoplasmic exoribonuclease Xrn1 in the budding yeast Saccharomyces cerevisiae, lacking RNA interference (RNAi). Whether the ribonuclease III Dicer affects aslncRNAs in close RNAi-capable relatives remains unknown. Using genome-wide RNA profiling, here we show that aslncRNAs are primarily targeted by the exosome and Xrn1 in the RNAi-capable budding yeast Naumovozyma castellii, Dicer only affecting Xrn1-sensitive lncRNAs (XUTs) levels in Xrn1-deficient cells. The dcr1 and xrn1 mutants display synergic growth defects, indicating that Dicer becomes critical in absence of Xrn1. Small RNA sequencing showed that Dicer processes aslncRNAs into small RNAs, with a preference for asXUTs. Consistently, Dicer localizes into the cytoplasm. Finally, we observed an expansion of the exosome-sensitive antisense transcriptome in N. castellii compared to S. cerevisiae, suggesting that the presence of cytoplasmic RNAi has reinforced the nuclear RNA surveillance machinery to temper aslncRNAs expression. Our data provide fundamental insights into aslncRNAs metabolism and open perspectives into the possible evolutionary contribution of RNAi in shaping the aslncRNAs transcriptome.
2019-08-21 | GSE90041 | GEO
Project description:Termination of RNAPII transcription is associated with RNA 3â end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in S. cerevisiae do not rely on RNA cleavage for termination, but instead terminate via a pathway that requires the Nrd1-Nab3-Sen1 (NNS) complex. Here we show that the S. pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination, but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3â end processing factors, is enriched at the 3â end of genes, and binds RNA motifs downstream of cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3â UTR length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3â end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 co-transcriptionally controls alternative polyadenylation. Two biological replicates of Seb1 and Control (parental strain) CRAC experiments
2016-07-22 | E-GEOD-82326 | biostudies-arrayexpress
Project description:To determine the effects of inactivation of both the nosense-mediated mRNA decay pathway and the general 5' to 3' decay pathway on yeast mRNA decay, we compared the expression profiles of the wild-type, xrn1, xrn1 upf1, xrn1 nmd2, and xrn1 upf3 strains.
2011-11-08 | E-MEXP-27 | biostudies-arrayexpress
Project description:Termination of RNAPII transcription is associated with RNA 3’ end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in S. cerevisiae do not rely on RNA cleavage for termination, but instead terminate via a pathway that requires the Nrd1-Nab3-Sen1 (NNS) complex. Here we show that the S. pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination, but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3’ end processing factors, is enriched at the 3’ end of genes, and binds RNA motifs downstream of cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3’ UTR length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3’ end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 co-transcriptionally controls alternative polyadenylation.
2016-07-22 | GSE82326 | GEO