Fig. 4
From: Roquin targets mRNAs in a 3′-UTR-specific manner by different modes of regulation
Nfkbid regulation requires Roquin to interact with at least three stem–loops. a Nucleotide sequences of the loop structures of wild-type mouse Nfkbid 3′-UTR stem–loops (SL) 1, 2, and 5 (gray) and introduced loop exchange mutations (LE: change of hexa- to tri-loops and vice versa, blue) are depicted. b, c Flow cytometry analysis of GFP in Rc3h1–2fl/fl; Cre-ERT2 MEF cells retrovirally transduced with the ICOS–GFP-Nfkbid 3′-UTR (1–263) reporter containing LE mutations (b) and individual or combined loop mutations (c) in the indicated stem–loops as described in Fig. 3b. MEF cells were either treated with 4′ OH-tamoxifen (iDKO) or left untreated (WT). MFI of GFP is shown for WT (red) and iDKO (blue) MEF cells. (d) Flow cytometry analysis of GFP in the λN-boxB-tethering system in Rc3h1–2−/− MEF cells stably expressing the reverse tetracycline-controlled transactivator rtTA3. MEF cells were first either transduced with a retrovirus containing the ICOS–GFP reporter with the wildtype Nfkbid 3′-UTR (1–263) or mutants containing one (at position of SL1, SL2, or SL5) or three (at the positions of SL1, SL2, and SL5) boxB structures. The cells were additionally transduced with a retrovirus encoding for λN-p2A-mCherry or λN-Roquin(K220A, K239A, and R260A)-p2A-mCherry containing a doxycycline-inducible cassette. Overexpression of the λN-constructs was induced by doxycycline administration for 14 h. MFI of GFP for WT (red) and iDKO (blue) MEF cells are indicated. e Fold-change of GFP MFI levels in cells as described in (d) with doxycycline-induced overexpression of λN-Roquin (K220A, K239A, and R260A)-p2A-mCherry, normalized to mean of wild-type Nfkbid (1–263). Error bars indicate mean ± SEM. Data are representative of six (b) and three (c–e) independent experiments
