Pumilio 2 controls translation by competing with eIF4E for 7-methyl guanosine cap recognition - PubMed (original) (raw)

Pumilio 2 controls translation by competing with eIF4E for 7-methyl guanosine cap recognition

Quiping Cao et al. RNA. 2010 Jan.

Abstract

Pumilio 2 (Pum2) interacts with the 3' UTR-containing pumilio binding element (PBE) of RINGO/SPY mRNA to repress translation in Xenopus oocytes. Here, we show that Pum2 also binds directly to the 5' 7mG cap structure; in so doing, it precludes eIF4E from binding the cap. Using deletion analysis, we have mapped the cap interaction domain of Pum2 to the amino terminus of the protein and identified a conserved tryptophan residue that mediates this specific interaction. Reporter mRNA-based assays demonstrate that Pum2 requires the conserved tryptophan to repress translation in injected Xenopus oocytes. Thus, in addition to its suggested role in regulating poly(A) tail length and mRNA stability, our results suggest that vertebrate Pumilio can repress translation by blocking the assembly of the essential initiation complex on the cap.

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Figures

FIGURE 1.

FIGURE 1.

Pum2 binds the cap. (A) Cytostatic Factor (CSF) extracts were prepared from Xenopus eggs and subsequently supplemented with calcium to induce entry into the cell cycle. At 0, 10, and 20 min after calcium addition, the extracts were supplemented with GTP and applied to m7G-Sepharose resin (lanes 1–3). In addition, some extract (no calcium) was supplemented with cap analog and also applied to the cap analog resin (lane 7). The proteins that were retained on the m7G-Sepharose resin were probed on Western blots for Maskin, Pum2, and eIF4E. The load fractions (i.e., 10% of total initial extract) were also probed for the same proteins (lanes 4–6). (B) Xenopus oocyte extracts were applied to GDP-Sepharose or m7G-Sepharose; the bound material was probed for Pum2 and eIF4E on a Western blot. The load fraction (10% of total) was also probed for the same proteins. (C) Reticulocyte lysates were primed with mRNA encoding Pum2 or eIF4E in the presence of 35S-methionine (lanes 3,4). Equal volumes of the lysates were mixed, supplemented with GTP or cap analog, and applied to m7G-Sepharose. Pum2 and eIF4E that were retained on the resin were detected by SDS-PAGE and phosphorimaging. (D) E. coli.-expressed Pum2 and eIF4E were applied to m7G-Sepharose or GDP-Sepharose columns; the bound material was examined by Western blotting (lanes 1–4). Ten percent of the load fractions were also probed by Western blotting (lanes 5,6). (E) Varying amounts of reticulocyte-synthesized Pum2 and eIF4E (i.e., μL of lysate) were applied to m7G-Sepharose resin in the presence of GTP or GTP plus cap analog and the amount retained was analyzed by Western blotting (top, bottom). In some cases, the lysates were mixed in the amounts indicated prior to being applied to m7G-Sepharose (bottom panel, lanes 1–5). In these cases, the lysates contained GTP but no free cap analog.

FIGURE 2.

FIGURE 2.

Pum2 W344 mediates cap binding and translational repression. (A) Pum2 deletion mutants and a W344G point mutant were expressed in reticulocyte lysates and applied to m7G-Sepharose; the percent bound was determined by Western blotting. (B) Sequence alignment of a selected region of Pum2 protein among animal species. The underlined W (corresponding to W344 in Xenopus laevis) is conserved among these and probably most other vertebrates as well. (C) Pum2 WT, Pum2 W344G, eIF4E, and ePAB were expressed in reticulocyte lysates, applied to GDP-Sepharose or m7G-Sepharose, and proteins that were retained were analyzed by Western blotting. (D) Recombinant Pum2 WT and W344G were applied to m7G-Sepharose and the bound material analyzed by Western blotting.

FIGURE 3.

FIGURE 3.

Pum2 represses translation in a W344-dependent manner. RNAs encoding Pum2 WT and W344G fused to λ N protein were injected into 10 oocytes. Following an overnight incubation, the oocytes were then injected with luciferase RNA containing 5 B boxes in the 3′ UTR. The oocytes were then homogenized and luciferase activity was determined (histogram) as was expression of the fusion proteins by Western blotting (bottom). The top portion shows an autoradiogram indicating that the relative levels of 32P-UTP trace-labeled luciferase RNAs at the end of the incubation period were similar. Other RNAs encoding Pum2 WT and W344G (no fusion) were injected into oocytes with luciferase RNA, whose 3′ UTR contained or lacked a PBE. Luciferase activity, expression of the heterologous proteins, and relative amount of the luciferase reporter RNAs that remained after the incubation period were determined as above. The amount of luciferase activity in the absence of heterologous Pum2 was used as the standard against which the other values were normalized. Each experiment was performed three times; the bars on the histograms refer to SEM. The data are statistically significant (P < 0.05, Student's _t_-test).

FIGURE 4.

FIGURE 4.

Pum2 and CPEB-mediated translational control. Comparison of the Pum2–cap interaction with the CPEB–Maskin–eIF4E interaction. The PBE refers to the pumilio binding element and the CPE to the cytoplasmic polyadenylation element.

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