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.

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.

Project description:Eukaryotic RNA polymerase II (Pol II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of the large subunit (Rpb1). Differential phosphorylation of Ser2, Ser5, and Ser7 in the 5M-bM-^@M-^Y and 3M-bM-^@M-^Y regions of genes coordinates the binding of transcription and RNA processing factors to the initiating and elongating polymerase complexes. Here, we report phosphorylation of Thr4 by Polo-like-kinase-3 in mammalian cells. ChIPseq analyses indicate an increase of Thr4-P levels in the 3M-bM-^@M-^Y region of genes occurring subsequently to an increase of Ser2-P levels. A Thr4/Ala mutant of Pol II displays a lethal phenotype. This mutant reveals a global defect in RNA elongation, while initiation is largely unaffected. Since Thr4 replacement mutants are viable in yeast we conclude that this amino acid has evolved an essential function(s) in the CTD of Pol II for gene transcription in mammalian cells. In this study, we investigated the function and ChIPseq genome-wide profiling of Thr4P residue (using the 6D7 antibody) of the Pol II CTD in Raji human B cells in comparison with either total Pol II profiling (N20 antibody, santa-cruz sc-899x), Ser5P CTD (3E8) or Ser2P (3E10) profiling in WT Raji cells. In another set of experiments, we also analysed total Pol II profiling (using an HA tag at the N-terminus of RPB1 and HA antibody Abcam ab9110) when endogenous enzyme is shut down by alpha-amanitin and replaced by either a recominant Pol II with 48 consensus repeats of the CTD (con48) or a mutated version where Thr4 residues were replaced by Ala (Thr4-Ala).In total 6 experimental sets (Pol IIt, Ser5P, Ser2P, Thr4P, con48, Thr4-Ala) were generated for our analysis and for each a biological replicate was performed. Biological replicates were merged when the data showed comparable signal noise ratio. Otherwise a unique replicate, showing the best noise ratio, was chosen for further analysis although the second replicate (for Ser2P and Thr4-Ala experiments). An input control (genomic DNA extracted after reverse crosslinking of the nuclear chip extracts) was performred and used for substraction to the ChIP experiments. One specific input material was used for wt cells, one for con48 and one for Thr4-Ala. Our data were processed to generate final wig files using our in house analysis pipeline essentially as described in Koch et al, (2011) NSMB 18 (8) p956.In brief, after alignment, sequence tags are: (i) artefact removed, (ii) elongated to an in silico optimized actual size of the initial fragments , (iii) input substracted, (iv) merged if applicable, (v) scaled for all experiments to correct for variation of tag number in between experiments. Several of the raw data files were no longer available.

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RNA polymerase II activity revealed by GRO-seq and pNET-seq in Arabidopsis

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