Cytoplasmic CstF-77 Protein Belongs to a Masking Complex with Cytoplasmic Polyadenylation Element-binding Protein in Xenopus Oocytes

Elisabeth Mandart,Catherine Papin,Christel Rouget

doi:10.1074/jbc.m601116200

Abstract

Regulated mRNA translation is a hallmark of oocytes and early embryos, of which cytoplasmic polyadenylation is a major mechanism. This process involves multiple protein components, including the CPSF (cleavage and polyadenylation specificity factor), which is also required for nuclear polyadenylation. The CstF (cleavage stimulatory factor), with CPSF, is required for the pre-mRNA cleavage before nuclear polyadenylation. However, some evidence suggests that the CstF-77 subunit might have a function independent of nuclear polyadenylation, which could be related to the cell cycle. As such, we addressed the question whether CstF-77 might have a role in cytoplasmic polyadenylation. We investigated the function of the CstF-77 protein in Xenopus oocytes, and show that CstF-77 has indeed a role in the cytoplasm. The Xenopus CstF-77 protein (X77K) localizes mainly to the nucleus, but also in punctuate cytoplasmic foci. We show that X77K resides in a cytoplasmic complex with eIF4E, CPEB (cytoplasmic polyadenylation element-binding protein), CPSF-100 and XGLD2, but is not required for cytoplasmic polyadenylation per se. Impairment of X77K function in ovo leads to an acceleration of the G(2)/M transition, with a premature synthesis of Mos and AuroraA proteins. However, the kinetic of Mos mRNA polyadenylation is not modified. Furthermore, X77K represses mRNA translation in vitro. These results suggest that X77K could be involved in masking of mRNA prior to polyadenylation.

Highlights

EXPERIMENTAL PROCEDURESX77K Cloning and Protein Alignment—A Xenopus leavis oocyte cDNA library in ␭gt from Clontech (ZL5000b) was screened with a 1.030-base pair (bp) probe obtained by RT-PCR from Xenopus RNA with the following degenerated oligonucleotides (Eurogentec): 5Ј-ATGGCYMARGCWTAYGAYTTYGCACT-3Ј and 5Ј-ACKCKSGTRTTRTTRTCYTCRTT-
The cleavage reaction requires two sequences, the highly conserved hexanucleotide AAUAAA located 30 –20 nucleotides upstream of the cleavage site, and the G/U-rich sequence that lies downstream of the 3Ј-cleavage site (DSE for downstream element)
CPEB directly binds to the CPE located upstream of the AAUAAA sequence, which is recognized by cleavage and polyadenylation specificity factor (CPSF), as in nuclear polyadenylation [14]

Summary

EXPERIMENTAL PROCEDURES

X77K Cloning and Protein Alignment—A Xenopus leavis oocyte cDNA library in ␭gt from Clontech (ZL5000b) was screened with a 1.030-base pair (bp) probe obtained by RT-PCR from Xenopus RNA with the following degenerated oligonucleotides (Eurogentec): 5Ј-ATGGCYMARGCWTAYGAYTTYGCACT-3Ј and 5Ј-ACKCKSGTRTTRTTRTCYTCRTT-. Oocyte extracts were incubated with 200 ng of antibodies over night at 4 °C on wheel, supplemented with 20 ␮l of protein A-Sepharose beads and mixed for another 45 min at 4 °C on wheel. Cultured cells were A Western blot analysis on oocyte and embryo lysates shows fixed using 4% paraformaldehyde (in PBS) for 15 min at room that X77K is expressed at a constant level during Xenopus temperature. In taining 1% Triton-X100 for 10 min at room temperature and S. cerevisiae and D. melanogaster, the CstF-77 homologues blocked in PBS containing 0.1% Tween and 3% BSA (blocking Rna and Su(f) respectively localize both to the nucleus and reagent).

RESULTS

We failed to detect endogenous

Findings

DISCUSSION