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. 2001 Oct;21(19):6359-68.
doi: 10.1128/MCB.21.19.6359-6368.2001.

Transcription-independent RNA polymerase II dephosphorylation by the FCP1 carboxy-terminal domain phosphatase in Xenopus laevis early embryos

B Palancade  1 , M F Dubois, M E Dahmus, O Bensaude
Affiliations

Transcription-independent RNA polymerase II dephosphorylation by the FCP1 carboxy-terminal domain phosphatase in Xenopus laevis early embryos

B Palancade et al. Mol Cell Biol. 2001 Oct.

Abstract

The phosphorylation of the RNA polymerase II (RNAP II) carboxy-terminal domain (CTD) plays a key role in mRNA metabolism. The relative ratio of hyperphosphorylated RNAP II to hypophosphorylated RNAP II is determined by a dynamic equilibrium between CTD kinases and CTD phosphatase(s). The CTD is heavily phosphorylated in meiotic Xenopus laevis oocytes. In this report we show that the CTD undergoes fast and massive dephosphorylation upon fertilization. A cDNA was cloned and shown to code for a full-length xFCP1, the Xenopus orthologue of the FCP1 CTD phosphatases in humans and Saccharomyces cerevisiae. Two critical residues in the catalytic site were identified. CTD phosphatase activity was observed in extracts prepared from Xenopus eggs and cells and was shown to be entirely attributable to xFCP1. The CTD dephosphorylation triggered by fertilization was reproduced upon calcium activation of cytostatic factor-arrested egg extracts. Using immunodepleted extracts, we showed that this dephosphorylation is due to xFCP1. Although transcription does not occur at this stage, phosphorylation appears as a highly dynamic process involving the antagonist action of Xp42 mitogen-activated protein kinase and FCP1 phosphatase. This is the first report that free RNAP II is a substrate for FCP1 in vivo, independent from a transcription cycle.

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Figures

FIG. 1
FIG. 1
CTD phosphatase activity in Xenopus. (A) Interphasic egg extracts or low-salt extracts from Xenopus A6 cells were incubated with labeled RNAP IIO for the amount of time indicated. (B) Extracts were incubated for 15 min with labeled RNAP IIO in the absence (control) or presence of 10 nM okadaic acid (OA), 5 nM microcystin (mic), or 120 mM KCl (K+). The labeled IIO form was incubated in pure buffer (–). Dephosphorylation of the CTD was visualized by SDS-PAGE followed by autoradiography. The positions of subunits IIa and IIo are indicated in both panels.
FIG. 2
FIG. 2
Alignment of FCP1 protein sequences. The human (Hs), Xenopus (Xl), and yeast (Sc) FCP1 sequences were deduced from their cDNA sequences (GenBank numbers AF154115, AF348120, and NC_001145). The homologies are in gray shaded boxes. The dark boxes enclose residues conserved in all three species. ◊, crucial aspartate residue.
FIG. 3
FIG. 3
X. laevis FCP1 exhibits CTD phosphatase activity. (A) Labeled RNAP IIO was incubated for 10 min with GST or increasing amounts of GST-xFCP1. (B) The experiment was carried out in the absence (control) or presence of 10 nM okadaic acid (OA), 5 nM microcystin (mic), or 120 mM KCl (K+). (C) Purified wild-type or mutant GST-xFCP1 fusion proteins were incubated for 10 min with labeled RNAP IIO. The amounts of the fusion proteins were checked by Western blot analysis with a GST-specific antibody. Dephosphorylation was visualized by SDS-PAGE and autoradiography. Unreacted RNAP IIO was loaded as a control (–) in each experiment. The positions of the subunits IIa and IIo are indicated in each panel.
FIG. 4
FIG. 4
Xenopus CTD phosphatase activity is attributable to xFCP1. (A) Low-salt A6 cell extract or purified GST-xFCP1wt fusion protein was analyzed by SDS-PAGE and Western blotting with anti-hFCP1 (α-hFCP1). Total lysates from control A6 cells or cells transfected with 5 μg of the plasmid encoding FLAG-xFCP1 were analyzed with anti-FLAG (α-FLAG). The positions of recombinant GST-xFCP1, endogenous xFCP1, and transfected FLAG-xFCP1 are indicated. (B) Labeled RNAP IIO was incubated for 15 min with crude egg or low-salt A6 cell extracts (control) which had been either mock or xFCP1 (α-FCP1) depleted. CTD dephosphorylation was monitored as indicated above. Nonincubated labeled RNAP IIO was loaded as a control (–). The positions of the subunits IIa and IIo are indicated. The presence of xFCP1 was checked by Western blotting.
FIG. 5
FIG. 5
CTD phosphorylation state after fertilization in Xenopus. (A) CTD dephosphorylation after fertilization in Xenopus embryos. Batches of 10 unfertilized eggs or embryos were sampled; lysed at 0 (unfertilized eggs), 1, 2, or 3 h postfertilization (hpf); and analyzed by SDS-PAGE along with a whole-cell lysate from A6 cells. The phosphorylation state of the largest RNAP II subunit was analyzed by Western blotting using an anti-Rpb1 antibody. The positions of the subunits IIa and IIo are indicated. (B) CTD dephosphorylation after in vitro activation of a meiotic extract. Activation was performed at 22°C by adding Ca2+ to the CSF-arrested meiotic extract for the indicated time. The phosphorylation state of the CTD and the level of active MAPK were analyzed by Western blotting with anti-Rpb1 and anti-active-MAPK (MAPK-P) antibodies, respectively. The positions of the subunits IIa and IIo are indicated. The presence of xFCP1 was checked by Western blot analysis.
FIG. 6
FIG. 6
CTD dephosphorylation upon activation of a meiotic extract requires xFCP1. (A) Experimental scheme. The CSF-arrested extract was immunodepleted for 30 min at 4°C before activation by Ca2+ at 22°C for the indicated time. Samples were taken at the various time points as indicated (arrows) and analyzed by Western blotting with the appropriate antibodies. (B) CTD phosphorylation, active MAPK (MAPK-P), and FCP1 were analyzed in untreated (U), mock-depleted (mock), and anti-FCP1-depleted (α-FCP1) extracts. The positions of the subunits IIa and IIo are indicated.
FIG. 7
FIG. 7
Rapid CTD phosphate turnover in CSF-arrested and interphasic egg extracts. (A) 32P-labeled (on the CTD repeats) RNAP IIO was added to CSF-arrested egg extract. Samples were analyzed after increasing incubation times by autoradiography and Western blotting. The positions of the subunits IIa and IIo are indicated. (B) 32P-labeled RNAP IIO was added to an egg extract that was either unactivated (▪) or activated (□) by Ca2+ at 22°C for 60 min. The percentage of radioactivity associated with subunit IIo (arbitrary units) is plotted as a function of time.
FIG. 8
FIG. 8
A model of two distinct CTD phosphorylation cycles. The FCP1 CTD phosphatase may counteract distinct CTD kinases in early embryos (A) or somatic cells (B).
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