Fig. 6

Roquin inhibits translation of Nfkbid. a mRNA, RPF and translation efficiency (TE) fold-change of ICOS–CDS alone or fused to the 3′-UTR of Nfkbid between untreated (WT) and 4′ OH-tamoxifen treated (iDKO) MEF cells to induce deletion of Roquin encoding alleles. b, d Representative polysome profiles of endogenous Nfkbid, Actb and Hprt, respectively, in Rc3h1-2^fl/fl; Cre-ERT2 MEF cells (b) as well as in Rc3h1–2^fl/fl; Cd4-Cre-ERT2 CD4⁺ T cells (d) treated with (iDKO) or without (WT) 4′ OH-tamoxifen. Cytoplasmic lysates from these cells were fractionated on sucrose gradients. The amounts of mRNA in each fraction were analyzed by RT-qPCR and are shown in percent of the sum detected in all fractions. One absorbance profile at 254 nm indicating the position of ribosomal subunits, ribosomes and polysomes for WT and iDKO MEF cells is shown in the top panel in (b). c, e The amounts of mRNA of Nfkbid, Actb, and Hprt in subpolysomal and polysomal fractions were determined from the polysome profiles as shown in (b, d). The subpolysomal and polysomal fractions were specified by the appropriate absorbance profile at 254 nm. Fractions 2–4 (c) or 2–5 (e) were defined as subpolysomal fractions and fractions 5–10 (c) or 6–10 (e) as polysomal fractions. The amounts of mRNA from these fractions were pooled and calculated in percent of the sum detected in all fractions. Statistical significance was determined by Wald test (a) and unpaired two-tailed Student’s t test (c, e); ns = not significant; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Error bars indicate mean ± SEM (a) or ± SD (c, d). Data are representative of three (a–c) and two (d, e) independent experiments