Figure 4.

POSH RING and SH3-3 domains are required for complementation of the POSH RNAi process outgrowth phenotype. (A) Domain structure of POSH and POSH mutants. RING: Really interesting new gene, a conserved feature of a subfamily of E3 ubiquitin ligases. SH3, Src homology 3 protein–protein interaction domain. RBD, Rac binding domain. (B and C) Complementation analysis in P19 neurons indicates a requirement for the POSH RING domain (B) and the POSH SH3-3 domain (C). P19 cells were transfected with Ngn2 and an RNAi complementation vector, which simultaneously allows for RNAi, complementation analysis, and marker expression from a single transcript. POSH6 RNAi targets the 3′-UTR of endogenous POSH, allowing for complementation by expression of the POSH coding sequence, which lacks 3′-UTR sequences. POSH6 RNAi+POSH denotes a vector that expresses POSH6 siRNAs, full-length POSH, and GFP. POSH6 RNAi+POSHΔRING denotes a vector that expresses POSH6 siRNAs, a POSH mutant deleted for the RING domain, and GFP. Vectors expressing full-length or various deletion mutants of POSH (shown in A) were transfected into P19 cells along with Ngn2. Neurons were fixed and stained for GFP, which marks the transfected neurons 70–75 h after transfection. Process length was measured on 100 cells per condition per experiment for two to three independent experiments. The distribution of process lengths (50–149, 150–249, and 250+ μm; left) as well as average process lengths (right) is shown. Pairwise comparisons that are statistically significant (Wilcoxon's rank sum test, left; Student's t test, right): control RNAi, POSH6 RNAi; control RNAi, POSH6RNAi+POSHΔRING; POSH6 RNAi, POSH6RNAi+POSH; POSH6 RNAi+POSH, POSH6RNAi+POSHΔ3/4; POSH6 RNAi, POSH6 RNAi+POSHΔ4.