Fig 1: A weakened BCCIP-eIF6 interaction impairs nucleolar recruitment of eIF6 and 60S ribosome biogenesis. (A) Co-precipitation between eIF6 and BCCIP mutants. A panel of mouse BCCIP mutants as indicated were fused with Flag-GFP, and transiently expressed in NIH3T3 cells. The cell extracts were collected and precipitated with M2 anti-Flag beads. The co-precipitated endogenous eIF6 and RPL23 were detected by Western blot. GFP alone (left lane) was used as a negative control. (B, C) Impaired in vitro interaction between mutated recombinant mouse BCCIP with eIF6. Wild type, L132P mutant, and the LL310/314AA mutant were co-expressed in BL21 cells with GST-eIF6. After IPTG induced protein expression, the cell lysates were pulled down with His- (B) or glutathione beads (C). The co-precipitated proteins were detected by western blots. (D–F) Rescue of growth defect by BCCIP mutants in BCCIP-depleted MEFs. EYFP tagged wild type, mutant L132P, or LL310/314AA were stably expressed in Bccipf/f;Rosa-CreERT2 cells respectively. EYFP alone was used as a control. Then the cells were treated with 4OHT to delete the endogenous Bccip gene, and used to determine polysome profiles (D), eIF6 localization (E) and the growth rates (F). In panel E, non-green Bccip-knockout cells were mixed with Bccip knockout cells expressing the EYFP-tagged exogenous Bccip, and stained for eIF6. Thus, all cells had depletion of endogenous BCCIP, but only the green cells had the expression of exogenous BCCIP proteins. As shown here, the expression of GFP-mBCCIP can enhance the localization of eIF6 to the nucleus (second column from left), while expression of EYFP, EYFP-L132P and EYFP-LL310/314 failed to enable the eIF6 recruitment in the nucleus after endogenous BCCIP depletion. In panel F. when comparing with EYFP control using Student t-test, the P-values among the 4OHT treated cells were: 0.0005 (wt. mBCCIP); 0.42 (mBCCIP-L132P) and 0.003 (mBCCIP-LL310/314AA). Among the ethanol treated cells, the P-values were: 0.56 (wt mBCCIP), P = 0.67 (mBCCIP-L132P) and P = 0.47 (mBCCIP-LL310/314AA).