Figure 6.

Competition of Roquin with AUF1 for the UCP3 ARE hub. (A) Top, schematic presentation of AUF1 domain arrangement with approximate position of the tandem RRM1-2 domains. Below that, the wild type UCP3 ARE hub is shown as used in this study and for the pulldown experiment in panel (B). (B) Analysis of Roquin binding to the UCP3 ARE hub. For RNA affinity purification, HEK293 whole cell lysates were incubated with the indicated UCP3 RNAs. AUF1 was visualized by Western blot using anti-AUF1 antibody. (C and D) NMR-based in vitro competition experiments between the Roquin ROQ domain and the AUF1 tandem RRM1-2. (C) Imino proton spectra of WT UCP3 CDE1 alone, in a 1:1 complex with AUF1 and after addition of increasing amounts of ROQ. (D) The same as in (C), but starting from a 1:1 complex of CDE1 with ROQ and subsequent titration of increasing amounts of AUF1 RRM1-2. For panels (C) and (D), the same spectral region is shown for ROQ alone for clarity towards protein-derived peaks. See also Supplementary Table S1 and Supplementary Figure S8.