Gene-specific RNA pol II phosphorylation and the "CTD code"

Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 10/2010; 17(10):1279-86. DOI: 10.1038/nsmb.1913
Source: PubMed


Phosphorylation of the RNA polymerase (Pol) II C-terminal domain (CTD) repeats (1-YSPTSPS-7) is coupled to transcription and may act as a 'code' that controls mRNA synthesis and processing. To examine the code in budding yeast, we mapped genome-wide CTD Ser2, Ser5 and Ser7 phosphorylations and the CTD-associated termination factors Nrd1 and Pcf11. Phospho-CTD dynamics are not scaled to gene length and are gene-specific, with highest Ser5 and Ser7 phosphorylation at the 5' ends of well-expressed genes with nucleosome-occupied promoters. The CTD kinases Kin28 and Ctk1 markedly affect Pol II distribution in a gene-specific way. The code is therefore written differently on different genes, probably under the control of promoters. Ser7 phosphorylation is enriched on introns and at sites of Nrd1 accumulation, suggesting links to splicing and Nrd1 recruitment. Nrd1 and Pcf11 frequently colocalize, suggesting functional overlap. Unexpectedly, Pcf11 is enriched at centromeres and Pol III-transcribed genes.

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    • "Available ChIP–chip data sets for Nrd1 and Pcf11 in wild-type cells and Pol II in pcf11-9 versus wild type from Kim et al. (2010) were reanalyzed and compared with analysis of newly generated ChIP-seq data (accession no. GSE67483). "
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    ABSTRACT: In Saccharomyces cerevisiae, short noncoding 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 also generally required for NRD-dependent transcription termination through the action of its C-terminal domain (CTD)-interacting domain (CID). Pcf11 localizes downstream from 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 restricted 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 from NRD terminators. We predict a mechanism by which the exchange of Nrd1 and Pcf11 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 Grzechnik et al.; Published by Cold Spring Harbor Laboratory Press.
    Genes & development 04/2015; 29(8):849-61. DOI:10.1101/gad.251470.114 · 10.80 Impact Factor
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    • "However, we cannot discard that the inhibition of transcription termination reported by excess of Npl3 (Bucheli and Buratowski 2005) could be one of the causes of this instability. Many transcription elongation and RNA processing factors, including THO–TREX, have been shown to be recruited along transcribed ORFs in a gradient profile that increases toward the 39 end of genes all over the genome (Kim et al. 2010; Mayer et al. 2010; Gomez-Gonzalez et al. 2011). This, together with the 39 end processing defects of THO mutants (Saguez et al. 2008) as well as the transcription termination role of Sen1 RNA–DNA helicase (Kawauchi et al. 2008), suggests that termination may be one of the most sensitive steps connected to RNA-mediated genome instability. "
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    • "The presence of Kin28 at promoters, the rapid reduction in Pol II occupancy upon Kin28 depletion, and the strong relationship between TBP occupancy and transcriptional activity (Kuras and Struhl, 1999; Li et al., 1999) suggest that Kin28 plays an important role in transcriptional initiation. Any role in transcriptional initiation would involve the kinase activity of Kin28, because a similar transcriptional defect is observed in the kin28-as mutant strain (Hong et al., 2009; Kim et al., 2010). "
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    ABSTRACT: The transition between transcriptional initiation and elongation by RNA polymerase (Pol) II is associated with phosphorylation of its C-terminal tail (CTD). Depletion of Kin28, the TFIIH subunit that phosphorylates the CTD, does not affect elongation but causes Pol II occupancy profiles to shift upstream in a FACT-independent manner indicative of a defect in promoter escape. Stronger defects in promoter escape are linked to stronger effects on preinitiation complex formation and transcription, suggesting that impairment in promoter escape results in premature dissociation of general factors and Pol II near the promoter. Kin28 has a stronger effect on genes whose transcription is dependent on SAGA as opposed to TFIID. Strikingly, Kin28 depletion causes a dramatic increase in Mediator at the core promoter. These observations suggest that TFIIH phosphorylation of the CTD causes Mediator dissociation, thereby permitting rapid promoter escape of Pol II from the preinitiation complex.
    Molecular cell 04/2014; 54(4). DOI:10.1016/j.molcel.2014.03.024 · 14.02 Impact Factor
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