Fig. 4: RBBP7 is incorporated into CRL4 and facilitates BUB3 ubiquitination.

a RepID dissociates from CRL4 during mitotic progression. HCT116 cells were transfected with RepID FL expression plasmid (for constructs, see Fig. 2b). Cells were collected at the indicated times after release from a nocodazole block, and chromatin-bound fractions were extracted. Immunoprecipitation was performed using anti-FLAG antibody, and co-precipitated proteins were analyzed by immunoblotting. b RBBP7 is incorporated into CRL4 after mitosis. Chromatin fractions of FLAG-DDB1-transfected HCT116 cells released from mitotic block at the indicated time points were immunoprecipitated with anti-FLAG antibody, followed by immunoblot analysis. c RBBP7, but not other DCAFs, localizes to mitotic spindle during mitosis. Immunofluorescence analysis was performed using the indicated anti-DCAFs antibodies in HCT116 WT cells. Scale bar: 10 μm. d RBBP7 colocalizes with BUB3 during metaphase. HCT116 RepID WT or KO cells were subject to immunofluorescence analysis using anti-RBBP7, BUB3, and anti-CREST antibodies with/without treatment with a p97/VCP inhibitor (CB5083). Zoomed-in areas (marked by white squares) are shown at the bottom of the composite images (merged images of the zoomed-in area are shown to the left of the single-color zoomed-in images). Scale bar: 10 μm. Quantification of colocalization between RBBP7 and BUB3 by Pearson’s correlation coefficients (bottom panel) (*p value < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant, Student’s t test). e Depletion of RepID-CRL4^RBBP7 components or BUB3 overexpression can delay mitotic exit. Depleted or overexpressed HCT116 cells were released from a nocodazole arrest for 3 h and analyzed by flow cytometry. Percentages of cells in subG1, G1, G2/M, and >4N phase are indicated. f RBBP7 facilitates the ubiquitination of BUB3 during mitosis. HIS-ubiquitin plasmids were transfected into HCT116 RepID WT cells together with siRNA-RBBP4 or si-RBBP7. Cells were synchronized, released, lysed, and proteins isolated on Ni-NTA beads as described in the legend for Fig. 3e. Immunoblot analysis was carried out using anti-BUB3 antibodies. g A model describing the DCAF switch on chromatin during mitosis. RepID recruits CRL4 on chromatin before metaphase, bringing CRL4 in contact with BUB3. RepID then dissociates from CRL4, and RBBP7 is incorporated into CRL4 as a catalytic DCAF for BUB3 ubiquitination.