Transcription-Independent RNA Polymerase II Dephosphorylation by the FCP1 Carboxy-Terminal Domain Phosphatase in Xenopus laevis Early Embryos

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 meioticXenopus 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 Xenopuseggs 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.

ACKNOWLEDGMENTS

This work was supported by grants from the Association pour la Recherche sur le Cancer (grant no. ARC 6250 to O.B.) and the Ligue Nationale Contre le Cancer (to O.B.).

We are much indebted to François Giudicelli, Jack Greenblatt, Olivier Jeanjean, Michael Kobor, and Daniela Marazziti for plasmids and reagents; to Olivier Hyrien and colleagues for Xenopushandling; and to Fréderic Gabriel and all members of the laboratory for help and discussions.