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:Drosophila contains one (dCDK12) and humans contain two (hCDK12 and hCDK13) proteins that are the closest evolutionary relatives of yeast Ctk1, the catalytic subunit of the major elongation-phase C-terminal repeat domain (CTD) kinase in Saccharomyces cerevisiae, CTDK-I. However, until now, neither CDK12 nor CDK13 has been demonstrated to be a bona fide CTD kinase. Using Drosophila, we demonstrate that dCDK12 (CG7597) is a transcription-associated CTD kinase, the ortholog of yCtk1. Fluorescence microscopy reveals that the distribution of dCDK12 on formaldehyde-fixed polytene chromosomes is virtually identical to that of hyperphosphorylated RNA polymerase II (RNAPII), but is distinct from that of P-TEFb (dCDK9 + dCyclin T). Chromatin immunoprecipitation (ChIP) experiments confirm that dCDK12 is present on the transcribed regions of active Drosophila genes. Compared with P-TEFb, dCDK12 amounts are lower at the 5' end and higher in the middle and at the 3' end of genes (both normalized to RNAPII). Appropriately, Drosophila dCDK12 purified from nuclear extracts manifests CTD kinase activity in vitro. Intriguingly, we find that cyclin K is associated with purified dCDK12, implicating it as the cyclin subunit of this CTD kinase. Most importantly, we demonstrate that RNAi knockdown of dCDK12 in S2 cells alters the phosphorylation state of the CTD, reducing its Ser2 phosphorylation levels. Similarly, in human HeLa cells, we show that hCDK13 purified from nuclear extracts displays CTD kinase activity in vitro, as anticipated. Also, we find that chimeric (yeast/human) versions of Ctk1 containing the kinase homology domains of hCDK12/13 (or hCDK9) are functional in yeast cells (and also in vitro); using this system, we show that a bur1(ts) mutant is rescued more efficiently by a hCDK9 chimera than by a hCDK13 chimera, suggesting the following orthology relationships: Bur1 ? CDK9 and Ctk1 ? CDK12/13. Finally, we show that siRNA knockdown of hCDK12 in HeLa cells results in alterations in the CTD phosphorylation state. Our findings demonstrate that metazoan CDK12 and CDK13 are CTD kinases, and that CDK12 is orthologous to yeast Ctk1.

Dataset Information

CTD kinase large subunit is encoded by CTK1, a gene required for normal growth of Saccharomyces cerevisiae.

Publications

CTD kinase large subunit is encoded by CTK1, a gene required for normal growth of Saccharomyces cerevisiae.

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