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Cytoplasmic polyadenylation-induced mRNA translation is a hallmark of early animal development. In Xenopus oocytes, where the molecular mechanism has been defined, the core factors that control this process include CPEB, an RNA binding protein whose association with the CPE specifies which mRNAs undergo polyadenylation; CPSF, a multifactor complex that interacts with the near-ubiquitous polyadenylation hexanucleotide AAUAAA; and maskin, a CPEB and eIF4E binding protein whose regulation of initiation is governed by poly(A) tail length. Here, we define two new factors that are essential for polyadenylation. The first is symplekin, a CPEB and CPSF binding protein that serves as a scaffold upon which regulatory factors are assembled. The second is xGLD-2, an unusual poly(A) polymerase that is anchored to CPEB and CPSF even before polyadenylation begins. The identification of these factors has broad implications for biological process that employ polyadenylation-regulated translation, such as gametogenesis, cell cycle progression, and synaptic plasticity.
Figure 1Interactions among CPEB, Symplekin, and CPSF. (A) Extracts from stage VI Xenopus oocytes were subjected to immunoprecipitation with CPEB antibody or a nonspecific IgG that served as a control (NB, all protein coimmunoprecipitation buffers contained RNase A). Western blots of the precipitated proteins were probed for symplekin and CPEB. Some oocytes were also injected with mRNA encoding myc-CPEB; myc antibody immunoprecipitates were probed for the myc epitope and symplekin, as were untreated extracts.
(B) Extracts from oocytes that had been incubated in the absence or presence of progesterone were subject to immunoprecipitation with symplekin antibody or control IgG. Western blots of both the immunodepleted extracts and the immunoprecipitated complexes were probed for symplekin, CPSF100, CPEB, and, as a negative control, mos.
(C) mRNAs encoding maskin and CPEB were translated in a reticulocyte lysate in the presence of 35S-methionine and applied to columns of S protein-agarose alone or S protein-agarose containing S-tagged recombinant symplekin. The columns were washed and the bound material analyzed by SDS-PAGE.
(D) 35S-methionine-labeled CPSF160 and symplekin were applied to columns containing S protein or S protein-CPEB and analyzed as in (C). Note that two proteins are produced by in vitro translation of symplekin mRNA; the faster migrating form could be due to downstream initiation, early termination, or protein breakdown.
Figure 2 Symplekin Is Required for Cytoplasmic Polyadenylation. (A) Oocytes were injected with symplekin antibody or a control IgG and incubated for 12–16 hr. A radiolabeled RNA probe containing or lacking a CPE was injected and the oocytes further incubated in the absence or presence of progesterone. The RNA was isolated and resolved on a 6% denaturing gel.
(B) Oocytes injected as in (A) were analyzed for symplekin and mos expression on Western blots.
(C) Egg extracts were mixed with increasing amounts of IgG or symplekin antibody and then primed with radiolabeled RNA probes containing or lacking a CPE. The RNA was analyzed as in (A).
Figure 3 Immunodepletion of Symplekin from Extracts Inhibits Polyadenylation
(A) Egg extracts were subjected to immunodepletion with symplekin antibody or two different control IgG antibodies. Some of the depleted extracts were supplemented with recombinant symplekin and then primed with CPE-containing RNA for an analysis of polyadenylation activity.
(B) Western blots of symplekin antibody-depleted extracts as well as the antibody-containing beads were probed for symplekin, CPSF100, and CPEB. A nonspecific band was recognized by the CPSF100 antibody.
(C) Extracts depleted with symplekin antibody or control IgG were probed for symplekin, CPSF100, and CPEB.
(D) The same extracts as those shown in (C) were supplemented with recombinant symplekin, symplekin plus recombinant CPEB, or these two proteins plus CPSF. The extracts were primed with CPE-containing RNA and examined for polyadenylation.
Figure 4 Identification of a Novel Xenopus Cytoplasmic Poly(A) Polymerase. (A) Representation of the 509 amino acid Xenopus GLD-2 (xGLD-2) protein, indicating the catalytic domain with a flanking conserved central domain region as well as the PAP/25A (polyA) polymerase) conserved domain and the nucleotidyltransferase domain. An alignment generated from ClustalW analysis of related GLD-2 sequences from several animals shows the conserved nucleotidyltransferase domain from C. elegans (ceGLD-2), human (hsGLD-2), mouse (mmGLD-2), and Xenopus (xGLD-2).
(B) Immunostaining of Xenopus XTC cells shows the cytoplasmic localization of xGLD-2. The cells were also immunostained for α-tubulin and costained with DAPI.
(C) Oocytes injected with mRNA encoding WT or catalytically inactive (D242A) XGLD-2 were also injected with CPE-containing RNA. Some oocytes were further treated with progesterone before being analyzed for polyadenylation.
Figure 5. xGLD-2 Interacts with the Cytoplasmic Polyadenylation Machinery and Promotes Polyadenylation.
(A) Extracts prepared from control or progesterone-matured oocytes were subject to immunodepletion with symplekin antibody or control IgG. Western blots of the antibody-containing beads were probed for symplekin, CPSF100, CPEB, and xGLD-2.
(B) Extracts depleted with symplekin antibody or control IgG as well as the antibody-containing beads were probed for symplekin, CPSF100, and xGLD-2.
(C) IgG and symplekin antibody-depleted extracts were supplemented with CPSF, symplekin, CPEB, and WT xGLD-2 or a xGLD-2 with a D242A substitution in the catalytic domain. The extracts were then primed with CPE-containing RNA and analyzed for polyadenylation.
(D) Antibody-containing beads from symplekin-depleted or IgG-depleted extracts were washed and mixed with CPE+ RNA, ATP, and PAP buffer and incubated for 1.5 hr. The RNA was then analyzed for polyadenylation, as was RNA from an undepleted extract.
(E) Symplekin and IgG-depleted extracts were supplemented with a mixture of CPSF, symplekin, and CPEB or a mixture of these proteins plus either WT xGLD-2 or a mutant xGLD-2 with a D242A mutation in the catalytic domain. The extracts were then analyzed for polyadenylation.
(F) Untreated extracts were supplemented with WT or D242A xGLD-2 protein and analyzed for polyadenylation of CPE-lacking or CPE-containing RNA.
(G) Untreated extracts were supplemented with WT xGLD-2 and analyzed for polyadenylation of RNA derived from a polylinker sequence (i.e., information-free), RNA containing an AAUAAA but lacking a CPE, or RNA containing both an AAUAAA and CPE.
Figure 6. Interactions within the Cytoplasmic Polyadenylation Complex.
(A) Recombinant xGLD-2 or GST, both S-tagged and bound to S protein-agarose beads, were incubated with 35S-methionine-labeled CPEB, symplekin, or CPSF160. The beads were washed and the proteins eluted in buffer containing 150 mM KCl, 250 mM KCl, or SDS. The proteins were resolved by 8% SDS-PAGE and visualized by phosphorimager analysis. Ten percent of the input of all three labeled proteins was analyzed in lane 1.
(B) S-tagged xGLD-2 bound to S protein-agarose was incubated with 35S-methionine-labeled CPEB proteins containing S174A/S180A (AA) or S174D/S180D (DD) double mutations. The beads were washed, and the bound proteins were analyzed as described above.
(C) Model of interactions among CPEB, CPSF, symplekin, and xGLD-2. P refers to phosphorylated CPEB serine 174.
