Fig. 8

Roquin regulates translation of targets with four or more binding sites. a Distribution of 3′-UTR lengths for Roquin-bound targets and nontargets. Boxes extend from the 25th to 75th percentiles (interquartile range (IQR)), horizontal lines represent the median, whiskers indicate the lowest and highest datum within 1.5*IQR from the lower and upper quartiles, respectively. b, c Cumulative distributions of mRNA and RPF (b), or TE (c) fold-change between Roquin-1 overexpressing (+dox) and Rc3h1–2^−/− (−dox) MEF cells. PAR-CLIP-identified targets were split based on the number of Roquin-binding sites that they contained (different colors). The comparison between the subsets of targets was performed with the Mann–Whitney U test and the P values for the two-tailed test are indicated. bs, binding site. Targets with 1 bs (n = 697), 2 bs (n = 161), 3 bs (n = 157) and 4+ bs (n = 40). Nontargets (n = 9322). (d, e) Reporter assays testing the full-length 3′-UTR of Sgk1 (c) as well as one individual part from the long 3′-UTR of Nfat5 (979-2630) (d) by flow cytometry showing untransduced Rc3h1–2^−/− MEF cells (dashed line) and transduced cells with (red line) or without (blue line) doxycycline-inducible expression of Roquin-1. MEF cells were treated with doxycycline for 14 h. MFI was calculated for GFP. (f) mRNA, RPF and TE fold-changes for the Roquin targets Sgk1 and Nfat5 in Roquin-1 overexpressing (+dox) compared to Rc3h1–2^−/− (−dox) MEF cells. (g, i) qPCR analysis of Sgk1 (g) and Nfat5 (i) gene expression in Rc3h1–2^−/− MEF cells treated with (+dox) or without (−dox) doxycycline for 14 h to induce Roquin-1 expression. (h, j) Automated capillary electrophoresis western analysis of SGK1 (h) and NFAT5 (j) in extracts of Rc3h1−2^−/− MEF cells treated with (+dox) or without (−dox) doxycycline for 14 h. Statistical significance was calculated by unpaired two-tailed Student’s t test (d, e, g–j) and Wald test (f); ns = not significant; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Error bars indicate mean ± SD (d, e, g–j) or ±SEM (f). Data are representative of 13 (a–c, f) and four (d, e, g–j) independent experiments