Comment[ArrayExpressAccession] E-GEOD-40254 MAGE-TAB Version 1.1 Public Release Date 2014-04-22 Investigation Title Ctk1 + CTD mutants Comment[Submitted Name] Ctk1 + CTD mutants Experiment 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. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Kemmeren Lenstra Tudek Libri Holstege Person First Name Patrick Tineke Agnieszka Domenico Frank Person Email geo@ncbi.nlm.nih.gov Person Affiliation UMC Utrecht Person Address Department of Molecular Cancer Research, UMC Utrecht, Universiteitsweg 100, Utrecht, Utrecht, Netherlands Person Roles submitter Protocol Name P-GSE40254-1 P-GSE40254-2 P-GSE40254-3 P-GSE40254-4 P-GSE40254-6 P-GSE40254-8 P-GSE40254-5 P-GSE40254-7 P-GSE40254-19 P-GSE40254-12 P-GSE40254-18 P-GSE40254-16 P-GSE40254-23 P-GSE40254-17 P-GSE40254-10 P-GSE40254-25 P-GSE40254-11 P-GSE40254-24 P-GSE40254-21 P-GSE40254-15 P-GSE40254-22 P-GSE40254-14 P-GSE40254-13 P-GSE40254-20 P-GSE40254-9 Protocol Description as genes, no bg corr, but followed by gene-specific dye bias correction. Identical to genes, no bg corr if the dye bias correction is 0.0, which is the default. So far (jan 2008), we have only dye-bias corrections for yeast. limma, A software package for the analysis of gene expression microarray data, especially the use of linear models for analysing designed experiments and the assessment of differential expression. Author(s): Gordon Smyth. The limma R package version 2.12.0 is used. P-values are Benjamini-Hochberg FDR corrected. ID_REF = VALUE = dye bias corrected normalized log2 ratio (Cy3/Cy5) Signal Norm_Cy5 = Signal Norm_Cy3 = as genes, no bg corr, but followed by gene-specific dye bias correction. Identical to genes, no bg corr if the dye bias correction is 0.0, which is the default. So far (jan 2008), we have only dye-bias corrections for yeast. limma, A software package for the analysis of gene expression microarray data, especially the use of linear models for analysing designed experiments and the assessment of differential expression. Author(s): Gordon Smyth. The limma R package version 2.12.0 is used. P-values are Benjamini-Hochberg FDR corrected. ID_REF = VALUE = dye bias corrected normalized log2 ratio (Cy5/Cy3) Signal Norm_Cy5 = Signal Norm_Cy3 = genes, no bg corr, dye corr limma ID_REF = VALUE = -INV_VALUE: dye bias corrected normalized log2 ratio (Cy3/Cy5) Signal Norm_Cy5 = Signal Norm_Cy3 = INV_VALUE = dye bias corrected normalized log2 ratio (Cy5/Cy3) genes, no bg corr, dye corr limma ID_REF = VALUE = dye bias corrected normalized log2 ratio (Cy5/Cy3) Signal Norm_Cy5 = Signal Norm_Cy3 = robot amplification and labeling v1.0 Tecan HS4800 hybridization yeast RNA isolation for robotic amplification v1.0 yeast HTP RNA isolation for robot v2.0 Yeast growth for expression profiling in Tecan platereader v1.13 Yeast growth for expression profiling in Tecan platereader v1.2 Yeast growth for expression profiling in Tecan platereader v1.12 Yeast growth for expression profiling in Tecan platereader v1.8 Yeast growth for expression profiling in Tecan platereader v1.17 Yeast growth for expression profiling in Tecan platereader v1.9 Yeast growth for expression profiling in Tecan platereader v1.0 Yeast growth for expression profiling in Tecan platereader v1.19 Yeast growth for expression profiling in Tecan platereader v1.1 Yeast growth for expression profiling in Tecan platereader v1.18 Yeast growth for expression profiling in Tecan platereader v1.15 Yeast growth for expression profiling in Tecan platereader v1.7 Yeast growth for expression profiling in Tecan platereader v1.16 Yeast growth for expression profiling in Tecan platereader v1.6 Yeast growth for expression profiling in Tecan platereader v1.3 Yeast growth for expression profiling in Tecan platereader v1.14 scanning of slides using Agilent G256BA Imagene feature extraction Protocol Type normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol labelling protocol hybridization protocol nucleic acid extraction protocol nucleic acid extraction protocol growth protocol growth protocol growth protocol growth protocol growth protocol growth protocol growth protocol growth protocol growth protocol growth protocol growth protocol growth protocol growth protocol growth protocol growth protocol growth protocol array scanning protocol Experimental Factor Name GENETIC MODIFICATION GENOTYPE Experimental Factor Type genetic modification genotype Publication Title The role of Ctk1 kinase in termination of small non-coding RNAs. Publication Author List Lenstra TL, Tudek A, Clauder S, Xu Z, Pachis ST, van Leenen D, Kemmeren P, Steinmetz LM, Libri D, Holstege FC PubMed ID 24324601 Publication DOI 10.1371/journal.pone.0080495 Comment[SecondaryAccession] GSE40254 Comment[GEOReleaseDate] 2014-04-22 Comment[ArrayExpressSubmissionDate] 2012-08-21 Comment[GEOLastUpdateDate] 2014-04-23 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-40254.sdrf.txt