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Incomplete RNA polymerase II phosphorylation in Xenopus laevis early embryos.
Phosphorylation of RNA polymerase II largest subunit on its C-terminal domain (CTD) heptapeptide repeats has been shown to play a key role in the regulation of mRNA synthesis and processing. In many higher metazoans, early embryos do not synthesise mRNAs during the first cell cycles following fertilisation. Transcription resumes and becomes an absolute requirement for development after several cell cycles characteristic of each species. Therefore, CTD phosphorylation has been investigated during early development of the African clawed-frog Xenopus laevis. Fertilisation is shown to trigger an abrupt dephosphorylation of the CTD. Phosphorylation of the CTD resumes concurrently with the mid-blastula transition (MBT). Both are advanced with polyspermy and increased temperatures; they do not occur when replication is impaired with aphidicolin. In Xenopus laevis somatic cells, a set of monoclonal antibodies defined distinct phosphoepitopes on the CTD. Two of them were absent before the MBT indicating that the CTD lacks the phosphorylation at the serine-2 position of the heptapeptide. The possible contribution of RNA polymerase II phosphorylation to the developmental-regulation of maternal mRNA processing in embryos is discussed.
Phosphorylation state of the RPB1 subunit in Xenopus embryos. Batches of ten unfertilised eggs (0) or embryos fertilised and allowed to develop at 16°C were sampled, lysed at the indicated time (hours post fertilisation, h.p.f.) and electrophoresed along with a whole lysate from A6 cells (A6). The RNAPII largest subunit was detected by western blot using the POL3/3 antibody. The positions of the IIa, IIo and IIe forms are indicated.
Influence of aphidicolin on the phosphorylation state of RPB1. Aphidicolin (100 μg/ml) was added (+) or not (−) at the two-cell stage and development was allowed to proceed at 25°C. Batches of ten unfertilised eggs (0) or embryos from the same female sampled respectively at 4.5, 6.5, 8.5 and 11.5 hours post fertilisation (h.p.f.) were analysed by western blot using the POL3/3 antibody. The positions of the IIa and the IIo forms are indicated.
The IIo appearance coincides with MBT. Embryos were allowed to develop at 23°C and analysed for RPB1 phosphorylation by western blot using the POL3/3 or the H5 antibody, or both. Batches of ten monospermic embryos (A) or polyspermic embryos (B) from the same female were sampled at the indicated hours post fertilisation (h.p.f.). The positions of the IIa, IIo and IIe forms are indicated.
Immunoreactivity of the RPB1 subunit in Xenopus embryos. Whole-cell lysates from A6 cells (A6), batches of ten eggs (Unfertilised) or embryos taken from the same female, maintained at 25°C and sampled at 4.5 (Pre-MBT), 6.5 (Post-MBT) or 8.5 (Gastrula) h.p.f. were analysed by western blot using the indicated monoclonal antibodies.
The IIm form of RPB1 is generated in serum-stimulated A6 cells. (A) Serum was added to quiescent A6 cells. Whole lysates (left) or lysates fractionated in a low-salt buffer into cytosolic supernatants (Sup) and pellets (Pel) from exponentially growing (Cex), quiescent (0) or serum-stimulated quiescent cells were analysed by western blot using the antibody POL3/3. The duration of the stimulation is indicated in minutes (min). (B) Whole lysates from exponentially growing A6 cells (Cex), cytosolic fraction from quiescent cells (0) or quiescent cells stimulated by serum during 10 minutes (10) were analysed by western blot. U0126 was added (lanes 4, 5) or not (lanes 2, 3) to culture medium 30 minutes before serum stimulation. The phosphorylation state of RPB1 and Xp42 was monitored using the POL3/3 and anti-ERK2 monoclonal antibodies (mAb), respectively. (C) Immunoreactivity of the IIm form. The samples from lanes 1 to 3 used in Fig. 5B were analysed by western blot with the indicated monoclonal antibodies. The positions of the IIa, IIo and IIm forms are indicated.