Figure 7.

PRPF8 regulates CP110 removal to promote ciliogenesis dependent on its ability to bind to the linear ubiquitin chains. (A) Schematic representation of the regions of PRPF8 binding to the linear ubiquitin chains (GST-Ub4). +, interaction; −, no interaction. (B) Map of the regions of PRPF8 binding to the linear ubiquitin chains (GST-Ub4). HEK293T cells were transfected with GFP-vector, or GFP-PRPF8 truncations. The cell lysates were pulled down by GST-Ub4, and then GFP proteins were detected by anti-GFP antibody. GST-Ub4 was stained by Ponceau S. (C) GFP-vector (−), GFP-PRPF8 WT, or ΔLUB (double depletions of residues 1301–1669 and 2234–2335 in full-length PRPF8) mutant were transfected into HEK293T cells. The subsequent GST pull-down assay procedures were similar to B. (D) GFP-vector (−), GFP-PRPF8 WT, or ΔLUB mutant were transfected into control or PRPF8-depleted RPE-1 cells. The cells were serum starved for 48 h and then stained with the indicated antibodies. Insets show zoomed-in views of the boxed regions. Scale bars, 5 µm (main image) and 1 µm (magnified region). (E) Quantification of cells with one CP110 dot in GFP-positive RPE-1 cells in D. (F) Quantification of the ciliated cells in GFP-positive RPE-1 cells in D. (G) The model of LUBAC regulating ciliogenesis. LUBAC specifically catalyzes the linear ubiquitination of the ciliary suppressor CP110. Subsequently, PRPF8 at the distal end of the mother centriole binds to the linear ubiquitin chains and facilitates the removal of the CP110–CEP97 complex from the mother centriole, therefore initiating ciliogenesis. Data are presented as mean ± SD of three independent experiments in E and F. **, P < 0.01; ***, P < 0.001 by one-way ANOVA with Bonferroni’s multiple comparisons test. FL, full-length; IB, immunoblot; tub, tubulin; Ub, ubiquitin.