Fig. 6.
Lengthening of the Gβγ-complex component GNB1 transcript is a central checkpoint in PCF11-TREND-mediated neuroblastoma differentiation. a PCF11 is significantly lower in low-risk compared to high-risk neuroblastoma patients (p = 0.005172; two-sided t-test; GSE4971044, ordered according to PCF11 expression; 10th percentile). b Low PCF11 in stage 4S neuroblastoma patients (compare Fig. 5g) is associated with pervasive TREND-lengthening (Supplementary Table 5; stage 4S, n = 48; stage 4, n = 65) including AES, IGF1R and GNB1. c GNB1 transcript lengthening correlates with a superior outcome in neuroblastoma patients (two-sided t-test). d GNB1 protein abundance (recorded by luciferase reporter assay) is determined by the GNB1 3′ UTR transcript isoform (middle; scheme of GNB1 3′ UTR luciferase reporter is shown in top). ‘Full’ and ‘Short’ denote 3′ UTR luciferase reporter constructs harbouring the (full-length) GNB1 3′ UTR processed at the distal or proximal polyadenylation signal (compare IGV track on the bottom before and after PCF11 depletion, for details see also legend to Fig. 2a). ‘Long’ indicates a construct with a 3′ UTR corresponding to sequences unique for the long transcript isoform (bar diagrams represent mean ± s.e.m. and a one-sided t-test p-value). e Depletion of the long GNB1 transcript isoform (siGNB1-L) in a low- or high-level PCF11 background (siRNA target sites highlighted in IGV tracks on top) results in reciprocal GNB1 mRNA isoform expression (middle panel) and protein output (lower panel) and counteracts a PCF11-KD-driven inhibition of WNT signalling (TCF7, compare Fig. 4c) and differentiation of neuroblastoma molecularly (TUBB3) and f phenotypically (cells are stained with an antibody directed against TUBB3 (red), nuclei are stained with DAPI (blue), scale bar = 100 µm). For box plots, centre line depicts median, hinges show 25th and 75th percentile, whiskers depict interquartile range (IQR = 1.5). *p < 0.05, **p < 0.01, ***p < 0.001