Investigating RNA polymerase II carboxyl‐terminal domain (CTD) phosphorylation

Abstract

Phosphorylation of RNA polymerase II's largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism. Numerous enzymes, including cell cycledependent kinases and TFIIF-dependent phosphatases target the CTD. However, the repetitive nature of the CTD prevents determination of phosphorylated sites by conventional biochemistry methods. Fortunately, a panel of monoclonal antibodies is available that distinguishes between phosphorylated isoforms of RNA polymerase II's (RNAP II) largest subunit. Here, we review how successful these tools have been in monitoring RNAP II phosphorylation changes in vivo by immunofluorescence, chromatin immunoprecipitation and immunoblotting experiments.The CTD phosphorylation pattern is precisely modified as RNAP II progresses along the genes and is involved in sequential recruitment of RNA processing factors. One of the most popular anti-phosphoCTD Igs, H5, has been proposed in several studies as a landmark of RNAP II molecules engaged in transcription. Finally, we discuss how global RNAP II phosphorylation changes are affected by the physiological context such as cell stress and embryonic development.Keywords: RNA polymerase II; CTD-phosphorylation; CTD-kinase; CTD-phosphatase; transcription; mRNA processing.The C-terminal domain (CTD) of RNA polymerase II's (RNAP II) largest subunit is essential for transcription [1][2][3], for its function as enhancer [4,5], for organization of transcription foci within the nucleus [6] and for pre-mRNA processing [7,8]. The CTD consists of multiple repeats of a seven amino acid motif [9,10] (Fig. 1). This motif has been conserved during evolution in eukaryotes, but the number of repeats varies depending on the species: 26 in yeast, 45 in flies and 52 in mammals. Five out of seven amino acids in the consensus motif are phosphate acceptors and indeed phosphorylation is a major post-translational modification of the CTD in vivo [11]. Serine O-glycosylation has also been reported as a minor modification with unknown functional significance [12]. In contrast, CTD phosphorylation plays a major role in the transcriptional process [13][14][15][16]. A large variety of kinases have been reported to phosphorylate the CTD of RNAP II in vitro [17,18]. Of particular significance, CDK7, CDK8 and CDK9 are subunits of the TFIIH general transcription factor, of the mediator complex and of the positive transcription elongation factor (P-TEFb), respectively.Interactions with the unphosphorylated CTD are involved in assembly of RNAP II with the mediator complex to form a holoenzyme or with general transcription factors to form a preinititiation complex of transcription. Phosphorylation of the CTD is required to disrupt these interactions at elongation of transcription and to assist the recruitment of pre-mRNA modification enzymes [16,19].Initial studies generally considered CTD phosphorylation as an all or nothing process. Here, we review recent evidence that multiple forms of CTD-phosphorylated RNAP II are present in cells. These forms have been character...

Embryo and Endosperm Inherit Distinct Chromatin and Transcriptional States from the Female Gametes in Arabidopsis

“…Transcriptional activity was followed using an antibody (H5; ab24758) raised against POLII that specifically targets the C-terminal domain of the main subunit of POLII when phosphorylated on Ser-2. As shown previously, the C-terminal domain phosphorylation pattern is modified as POLII engages in transcript elongation (Palancade and Bensaude, 2003). The H5 antibody has thus been proposed as a landmark of transcriptional activity.…”

“…To verify the relation between POLII downregulation and the resulting phenotype, we used an antibody (H5) directed against the active isoform of the main subunit of POLII. The H5 antibody specifically recognizes the heptamer of the POLII C-terminal domain when phosphorylated on Ser-2, which is a hallmark of POLII engaged in transcript elongation (Palancade and Bensaude, 2003). In these RNAi lines, abnormal seeds showed no detectable active POLII as determined by indirect immunolocalization (see Supplemental Figure 1D and Supplemental Table 1 online), strongly suggesting that the arrest was a phenotypic response to the downregulation of POLII.…”

Alteration of cyclin D1 transcript elongation by a mutated transcription factor up-regulates the oncogenic D1b splice isoform in cancer

“…C-ter domain of the Rpb1 subunit of Pol II (CTD) on Ser-5 residues, which is specifically recognized by the H14 antibody; in contrast, the CTD4H8 antibody recognizes both phosphorylated and unphosphorylated forms of Pol II (28). Remarkably, depletion of EWS-FLI1 induced a 2-fold decrease in Ser-5 phosphorylation levels but did not decrease Pol II levels at the cyclin D1 gene 5Ј end (Fig.…”

Major Histocompatibility Class II Transactivator (CIITA) Mediates Repression of Collagen (COL1A2) Transcription by Interferon γ (IFN-γ)

“…Second, since CIITA is an acetyltransferase (51), once recruited to the gene, the enzyme could be activated and modify RNA Pol II or proteins in the holoenzyme complex. The C-terminal domain of RNA Pol II is a substrate for various modifications, which alter preinitiation complex formation, promoter clearance, elongation and splicing (52)(53)(54)(55)(56). Recent evidence suggests that CIITA recruitment to MHC-II promoter correlates with phosphorylation of serine 5 within the C-terminal domain of RNA Pol II (48).…”