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

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.