Transcribing RNA polymerase II is phosphorylated at CTD residue serine-7

Institute of Clinical Molecular Biology and Tumour Genetics, GSF-Research Center of Environment and Health, Munich Center for Integrated Protein Science (CiPSM), Marchioninistrasse 25, 81377 Munich, Germany.
Science (Impact Factor: 33.61). 01/2008; 318(5857):1780-2. DOI: 10.1126/science.1145977
Source: PubMed


RNA polymerase II is distinguished by its large carboxyl-terminal repeat domain (CTD), composed of repeats of the consensus
heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Differential phosphorylation of serine-2 and serine-5 at the 5′ and 3′ regions of genes appears to coordinate the localization
of transcription and RNA processing factors to the elongating polymerase complex. Using monoclonal antibodies, we reveal serine-7
phosphorylation on transcribed genes. This position does not appear to be phosphorylated in CTDs of less than 20 consensus
repeats. The position of repeats where serine-7 is substituted influenced the appearance of distinct phosphorylated forms,
suggesting functional differences between CTD regions. Our results indicate that restriction of serine-7 epitopes to the Linker-proximal
region limits CTD phosphorylation patterns and is a requirement for optimal gene expression.

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    • "Unspecific binding of antibodies was blocked by incubation with 5% milk in TBS-T before being incubated with primary antibodies diluted in blocking solution. The primary antibodies we used are as follows: a-Rpb1 (POL 3/3); a-HA (3F10, Roche); CTD phospho-specific antibodies a-Y 1 -P, S 2 -P, T 4 -P, S 5 -P, and S 7 -P (5G9, 3D12, 3E10, 6D7, 3E8, and 4E12) (Chapman et al., 2007); and a-tubulin (T6557) (Sigma). Afterward, the membranes were either incubated with IRDye-labeled, secondary antibodies against rat (680 nm; Alexa, Invitrogen) and mouse (800 nm; Rockford, Biomol) and analyzed using an Odyssey Imaging System (Li-Cor) or they were stained with HRP-conjugated secondary antibodies against rat (Sigma) or mouse (Promega) and revealed by enhanced chemiluminescence. "
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    ABSTRACT: The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5, and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD.
    Full-text · Article · Jan 2016 · Molecular Cell
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    • "RNAPII- 8WG16 (MMS-126R) antibody ( Jones et al., 2004) was purchased from Covance. RNAPII-Ser5P (clone 3E8, 04-1572) antibody (Chapman et al., 2007) was purchased from Millipore and RNAPII-Ser5P (ab5131) antibody (Rahl et al., 2010) was purchased from Abcam. H3K4me1 (ab8895), H3K4me3 (ab1012), H3K9ac (ab4441), H3K27ac (ac4729), H3K36me3 (ab9050), and H3K79me2 (ab3594) antibodies were purchased from Abcam. "
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    ABSTRACT: Genome-wide analyses have revolutionized our ability to study the transcriptional regulation of circadian rhythms. The advent of next-generation sequencing methods has facilitated the use of two such technologies, ChIP-seq and RNA-seq. In this chapter, we describe detailed methods and protocols for these two techniques, with emphasis on their usage in circadian rhythm experiments in the mouse liver, a major target organ of the circadian clock system. Critical factors for these methods are highlighted and issues arising with time series samples for ChIP-seq and RNA-seq are discussed. Finally, detailed protocols for library preparation suitable for Illumina sequencing platforms are presented. © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Feb 2015 · Methods in enzymology
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    • "The DYRK1A preference for Ser2p and Ser5p is in agreement with DYRK1A favoring Pro residues at the +1 position of its phosphorylation motif (Himpel et al., 2000). Notably, the DYRK1A-phosphorylated CTD was also detected with the H5 antibody (Figure S6E), which has greater affinity for CTD phosphorylated at both Ser2 and Ser5 rather than Ser2 alone (Chapman et al., 2007), suggesting that DYRK1A could phosphorylate both residues within the same repeat, at least in vitro. Moreover, the large band shift observed upon phosphorylation indicates that DYRK1A can phosphorylate several of the hepta-repeats in the same molecule, as it has been described for the CTD kinase CDK9 (Czudnochowski et al., 2012). "
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    ABSTRACT: DYRK1A is a dosage-sensitive protein kinase that fulfills key roles during development and in tissue homeostasis, and its dysregulation results in human pathologies. DYRK1A is present in both the nucleus and cytoplasm of mammalian cells, although its nuclear function remains unclear. Genome-wide analysis of DYRK1A-associated loci reveals that the kinase is recruited preferentially to promoters of genes actively transcribed by RNA polymerase II (RNAPII), which are functionally associated with translation, RNA processing, and cell cycle. DYRK1A-bound promoter sequences are highly enriched in a conserved palindromic motif, which is necessary to drive DYRK1A-dependent transcriptional activation. DYRK1A phosphorylates the C-terminal domain (CTD) of RNAPII at Ser2 and Ser5. Depletion of DYRK1A results in reduced association of RNAPII at the target promoters as well as hypophosphorylation of the RNAPII CTD along the target gene bodies. These results are consistent with DYRK1A being a transcriptional regulator by acting as a CTD kinase. Copyright © 2015 Elsevier Inc. All rights reserved.
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