Fig. 5

Nfkbid regulation by Roquin redundantly involves decapping and deadenylation. a Schematic representation of ICOS reporter constructs with or without 5′ cap (m₇G) and poly(A) tail (polyA). (b) Flow cytometry analysis of ICOS reporter expression in Rc3h1–2^−/− MEF cells containing a doxycycline-inducible cassette for re-expression of Roquin-1. Rc3h1–2^−/− MEF cells were treated with doxycycline (+dox) for 16–20 h or left untreated (−dox) and electroporated with the different reporter constructs as indicated in (a). c, e, g, i Schematic representation of reporter constructs composed of the ICOS–CDS fused to the 3′-UTR of Nfkbid. The 5′ end of each reporter construct either contained a 5′ cap (m₇G), ARCA (anti-reverse cap), S-D1, S-D2, or BH₃ cap analogs (c, e, g, i). The 3′ end either contained a poly(A) tail consisting of 146 adenine bases (polyA₁₄₆) (c, e, g, i) or a polyA₁₄₆ tail modified by the addition of ten cytosines (C₁₀) to block deadenylation (c, g, i). d, f, h Flow cytometry analysis of ICOS reporter expression in Rc3h1–2^−/− MEF cells containing a doxycycline-inducible cassette for re-expression of Roquin-1. Rc3h1–2^−/− MEF cells were treated with doxycycline (+dox) for 16–20 h or left untreated (−dox) and electroporated with the different reporter constructs as indicated in (c, e, g). Black lines represent nonelectroporated cells. j Quantification of ICOS reporter expression using flow cytometry in Rc3h1–2^−/− MEF cells transfected with Roquin relative to cells without Roquin transfection. These cells contain a doxycycline-inducible shRNA against cnot1 and were treated with doxycycline for 3 days before electroporation with the different reporter constructs as indicated in (i). cNOT1 knock-down (KD) samples represent only GFP positive and therefore shRNA expressing cells. k Immunoblot analysis of knockdown of cNOT1 expression in MEF cells containing a doxycycline-inducible shRNA against cnot1. Only GFP⁺ cells express the shRNA. A nontargeting (NT) shRNA was used as a control. Statistical significance was determined by unpaired two-tailed Student’s t test (j), ns = not significant; ^*p < 0.05; ^**p < 0.01. Error bars represent three technical replicates (mean ± SEM). Data are representative of one (b, k), seven (d), two (h, j), and three (f) independent experiments