Figure 5: Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR.
From: Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40
(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).
