Fig 2: BCCIP regulates 60S subunit biogenesis by modulating pre-rRNA processing. (A, B) Relative abundance of BCCIP and representative ribosomal proteins in mature (A) and pre-ribosome (B) fractions. MEFs were collected, and the cytoplasmic extracts (A) and pre-ribosome-containing nuclear extracts (B) were separated by sedimentation through sucrose gradients. Fractions were concentrated to an equal volume and analyzed by Western blotting with the antibodies for RPS3, RPL22, RPL23a, eIF6 and BCCIP. The lanes in the Western blots are aligned with the gradient fractions. The left and right lanes were loaded with whole-cell extract to assist the identification of the protein species and provide a relative abundance reference in the Western blot. (C) Illustration of the major pre-ribosomal RNA species (pre-rRNAs) in human and mouse cells. Positions of hybridization probes (p1-p4) relative to cleavage sites in pre-rRNA are shown at the top. See Supplementary Table S1 for the sequence of the probes. (D, E) Requirement of BCCIP for pre-rRNA maturation. Bccipwt/wt;Rosa-CreERT2 MEFs and Bccipf/f;Rosa-CreERT2 MEFs were treated with 4OHT for the indicated lengths of days (D). The HT1080 cells capable of Dox-induced BCCIP knockdown were treated with Dox for the indicated time (E). The cells were collected and their RNA was analyzed by Northern hybridization with probe p4. d.p., degradation products of the pre-rRNA. See Supplementary Figures S15 and S16 for hybridizations with additional probes.