The Nrd1 CTD-interaction domain contributes to the choice of RNA polymerase II termination pathway and couples to RNA processing by the nuclear exosome
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Michael Lidschreiber
PROVIDER: E-MTAB-1626 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:ChIP-chip was performed to identify the genomic binding locations for the termination factors Nrd1, and Rtt103, and for RNA polymerase (Pol) II phosphorylated at the tyrosine 1 and threonine 4 position of its C-terminal domain (CTD). In different phases of the transcription cycle, Pol II recruits different factors via its CTD, which consists of heptapeptide repeats with the sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Here we show that the CTD of transcribing yeast Pol II is phosphorylated at Tyr1, and that this impairs recruitment of termination factors. Tyr1 phosphorylation levels rise downstream of the transcription start site (TSS), and decrease before the polyadenylation (pA) site. Tyr1-phosphorylated gene bodies are depleted of CTD-binding termination factors Nrd1, Pcf11, and Rtt103. Tyr1 phosphorylation blocks CTD binding by these termination factors, but stimulates binding of elongation factor Spt6. These results show that CTD modifications can not only stimulate but also block factor recruitment, and lead to an extended CTD code for transcription cycle coordination.
2012-06-29 | E-MTAB-1060 | biostudies-arrayexpress
Project description:The Nrd1-Nab3-Sen1 (NNS) complex plays a pivotal role in the control of pervasive transcription and the generation of sn- and snoRNAs in S. cerevisiae. The NNS-complex terminates transcription of non-coding RNA genes and promotes processing/degradation of transcripts by the nuclear exosome. To assess the role of the Nrd1p CTD-interacting domain (CID) in the function of the NNS-complex, we re-examined whether this domain is required for efficient transcription termination by the NNS pathway. We compared the RNA polymerase II distribution by ChIP and tiling arrays (ChIP-chip) in wild type and nrd1 deltaCID cells. Moreover, we compared the genome-wide chromatin distribution of Nrd1p in the presence and the absence of the CID by ChIP-chip analysis. ChIP of RNA polymerase II was performed using an anti-Rpb3 antibody (1Y26, Neoclone). ChIP of Nrd1 was performed using TAP-tagged S. cerevisiae strains. For details see protocols. To download the wild-type Pol II and Nrd1 data go to E-MTAB-1626 and E-MTAB-1060, respectively.
2014-04-24 | E-MTAB-2175 | biostudies-arrayexpress
Project description:At the 3'-ends of genes, RNA polymerase (Pol) II is dephosphorylated at tyrosine 1 residues of its C-terminal domain (CTD), resulting in recruitment of transcription termination factors. We show that the multisubunit cleavage and polyadenylation factor (CPF) is a Pol II CTD phosphatase and its Glc7 subunit is required for Tyr1 dephosphorylation at the poly-adenylation site and Pol II termination in vivo. ChIP-chip was performed to examine the effect of Glc7 nuclear depletion on genome-wide Pol II occupancy [using ?-Rpb3 (1Y26, cat. no. W0012, neoclone) antibody] and CTD tyrosine 1 phosphorylation levels [using ?-TyrY1P (3D12, D. Eick) antibody].
2013-12-16 | E-MTAB-1528 | biostudies-arrayexpress
Project description:In Saccharomyces cerevisiae short non-coding RNA (ncRNA) generated by RNA Polymerase II (Pol II) are terminated by the NRD complex consisting of Nrd1, Nab3 and Sen1. We now show that Pcf11, a component of the cleavage and polyadenylation complex (CPAC), is generally required for NRD-dependent transcription termination through the action of its CTD interacting domain (CID). Pcf11 localizes downstream of Nrd1 on NRD terminators, and its recruitment depends on Nrd1. Furthermore mutation of the Pcf11 CID results in Nrd1 retention on chromatin, delayed degradation of ncRNA and restricts Pol II CTD Ser2 phosphorylation and Sen1-Pol II interaction. Finally, the pcf11-13 and sen1-1 mutant phenotypes are very similar as both accumulate RNA:DNA hybrids and display Pol II pausing downstream of NRD terminators. We predict a mechanism whereby Nrd1 and Pcf11 exchange on chromatin facilitates Pol II pausing and CTD Ser2-P phosphorylation. This in turn promotes Sen1 activity that is required for NRD-dependent transcription termination in vivo. ChIP-seq with antibody against pol II in wild type and Pcf11 mutants: Pcf11-2, Pcf11-9 and Pcf11-13 grown at 25C and 37C along with input samples
2015-04-02 | E-GEOD-67483 | biostudies-arrayexpress
Project description:In Saccharomyces cerevisiae short non-coding RNA (ncRNA) generated by RNA Polymerase II (Pol II) are terminated by the NRD complex consisting of Nrd1, Nab3 and Sen1. We now show that Pcf11, a component of the cleavage and polyadenylation complex (CPAC), is generally required for NRD-dependent transcription termination through the action of its CTD interacting domain (CID). Pcf11 localizes downstream of Nrd1 on NRD terminators, and its recruitment depends on Nrd1. Furthermore mutation of the Pcf11 CID results in Nrd1 retention on chromatin, delayed degradation of ncRNA and restricts Pol II CTD Ser2 phosphorylation and Sen1-Pol II interaction. Finally, the pcf11-13 and sen1-1 mutant phenotypes are very similar as both accumulate RNA:DNA hybrids and display Pol II pausing downstream of NRD terminators. We predict a mechanism whereby Nrd1 and Pcf11 exchange on chromatin facilitates Pol II pausing and CTD Ser2-P phosphorylation. This in turn promotes Sen1 activity that is required for NRD-dependent transcription termination in vivo.
2015-04-02 | GSE67483 | GEO
Project description:Transcription termination in Saccharomyces cerevisiae can be performed by at least two distinct pathways and is directed by the phosphorylation status of the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II). Late termination of mRNAs is performed by the CPF/CF complex and requires CTD-Ser2 phosphorylation. Early termination of shorter cryptic unstable transcripts (CUTs) and small nucleolar RNAs (snoRNAs) is preformed by the Nrd1 complex, and requires CTD-Ser5 phosphorylation. In this study, mutants of the different termination pathways were compared by genome-wide expression analysis. Surprisingly, the expression changes observed upon loss of the CTD-Ser2 kinase Ctk1 are more similar to loss of a subunit of the Ser5P binding Nrd1-complex, than to loss of Ser2P binding factors. Tiling array analysis of ctk1Δ reveals readthrough at several hundred sites, including snoRNAs, as reported previously, but also many cryptic unstable transcripts, stable untranslated transcripts (SUTs) and other transcripts. Surprisingly, neither loss of CTK1 nor a Pol II CTD-Ser2 substitution mutant results in a global defect in termination of mRNAs, indicating that Ser2P is not essential for proper termination of most mRNAs. At snoRNA, Nrd1 location is shifted downstream in ctk1∆, indicating defective release rather than recruitment of Nrd1. Weakening the interaction between Nrd1 and Pol II rescues the readthrough in ctk1∆, likely by facilitating Nrd1 release. The termination defect is kinase activity dependent, but cannot be completely explained by loss of CTD-Ser2 phosphorylation , a major substrate of Ctk1, suggesting the involvement of an additional substrate. Mutant alleles of the elongation factor Spt5 rescue ctk1∆-dependent readthrough, indicating a role for Spt5 in this process, perhaps as a substrate of Ctk1. The results show that Ctk1 is more intimately involved in termination of small non-coding RNAs than was previously assumed and lead to a model in which Ctk1 influences Spt5 activity to achieve this.
2014-04-22 | GSE40254 | GEO
Project description:Bypass of Ess1 (Bye1) is a nuclear protein with a domain resembling the central domain in the transcription elongation factor TFIIS. Bye1 binds with its TFIIS-like domain (TLD) to RNA polymerase (Pol) II. Using a Bye1-TAP strain ChIP-chip was performed to examine the genome-wide association of Bye1 with chromatin in vivo. Bye1 is recruited to chromatin via its TLD and occupies the 5'-region of active genes. A plant homeo domain (PHD) in Bye1 binds histone H3 tails with trimethylated lysine 4, and this interaction is enhanced by the presence of neighboring posttranslational modifications (PTMs) that mark active transcription and conversely is impaired by repressive PTMs. We identify putative human homologs of Bye1, the proteins PHD finger protein 3 and death-inducer obliterator, which are both implicated in cancer. These results establish Bye1 as the founding member of a unique family of chromatin transcription factors that link histones with active PTMs to transcribing Pol II.
2013-09-23 | E-MTAB-1894 | biostudies-arrayexpress
Project description:Genome-wide studies have identified abundant small, non-coding RNAs including snRNAs, snoRNAs, cryptic unstable transcripts (CUTs), and upstream regulatory RNAs (uRNAs) that are transcribed by RNA polymerase II (pol II) and terminated by a Nrd1-dependent pathway. Here, we show that the prolyl isomerase, Ess1, is required for Nrd1-dependent termination of ncRNAs. Ess1 binds the carboxy terminal domain (CTD) of pol II and is thought to regulate transcription by conformational isomerization of Ser-Pro bonds within the CTD. In ess1 mutants, expression of ~10% of the genome was altered, due primarily to defects in termination of snoRNAs, CUTs, SUTs and uRNAs. Ess1 promoted dephosphorylation of Ser5 (but not Ser2) within the CTD, most likely by the Ssu72 phosphatase, and we provide evidence for a competition between Nrd1 and Pcf11 for CTD-binding that is regulated by Ess1-dependent isomerization. This is the first example of a prolyl isomerase required for interpreting the “CTD code.”
2009-10-16 | GSE17638 | GEO
Project description:Transcription termination in Saccharomyces cerevisiae can be performed by at least two distinct pathways and is directed by the phosphorylation status of the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II). Late termination of mRNAs is performed by the CPF/CF complex and requires CTD-Ser2 phosphorylation. Early termination of shorter cryptic unstable transcripts (CUTs) and small nucleolar RNAs (snoRNAs) is preformed by the Nrd1 complex, and requires CTD-Ser5 phosphorylation. In this study, mutants of the different termination pathways were compared by genome-wide expression analysis. Surprisingly, the expression changes observed upon loss of the CTD-Ser2 kinase Ctk1 are more similar to loss of a subunit of the Ser5P binding Nrd1-complex, than to loss of Ser2P binding factors. Tiling array analysis of ctk1Δ reveals readthrough at several hundred sites, including snoRNAs, as reported previously, but also many cryptic unstable transcripts, stable untranslated transcripts (SUTs) and other transcripts. Surprisingly, neither loss of CTK1 nor a Pol II CTD-Ser2 substitution mutant results in a global defect in termination of mRNAs, indicating that Ser2P is not essential for proper termination of most mRNAs. At snoRNA, Nrd1 location is shifted downstream in ctk1∆, indicating defective release rather than recruitment of Nrd1. Weakening the interaction between Nrd1 and Pol II rescues the readthrough in ctk1∆, likely by facilitating Nrd1 release. The termination defect is kinase activity dependent, but cannot be completely explained by loss of CTD-Ser2 phosphorylation , a major substrate of Ctk1, suggesting the involvement of an additional substrate. Mutant alleles of the elongation factor Spt5 rescue ctk1∆-dependent readthrough, indicating a role for Spt5 in this process, perhaps as a substrate of Ctk1. The results show that Ctk1 is more intimately involved in termination of small non-coding RNAs than was previously assumed and lead to a model in which Ctk1 influences Spt5 activity to achieve this. Two channel microarrays were used. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used in one of the channels for each hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Using the Erlenmeyer growth protocol up to five deletion strains were grown on a single day. In the tecan platereader, up to eleven deletion strains could be grown on a single day. Wt cultures were grown parallel to the deletion mutants to assess day-to-day variance.
2014-04-22 | E-GEOD-40254 | biostudies-arrayexpress
Project description:Termination of Pol II transcription is an important step in the transcription cycle and is responsible for dislodgement of polymerase from DNA which leads to the release of a functional transcript. Recent studies have identified key players important for termination and showed a conserved domain that interacts with the phosphorylated C-terminus of Pol II (CTD-Interacting-Domain, CID) to constitute a common feature of these proteins. However, the mechanism by which transcription termination is achieved, is not understood. Using genome-wide methods, we demonstrate that the fission yeast CID protein Seb1 is essential for termination of protein-coding and non-coding genes through interacting with S2-phosphorylated Pol II and nascent RNA. Furthermore, we present the crystal structures of the Seb1 CTD- and RNA-binding modules. Unexpectedly, the latter reveals a novel intertwined two-domain arrangement of a canonical RRM and a second domain. These results provide important insights into the mechanism underlying eukaryotic transcription termination.
2017-02-05 | GSE89914 | GEO