Fig. 3. A fragment of RORC 3ʹUTR forms an ensemble of two alternative structures.

a, Arc representation of the two alternative conformations of the RORC RNA switch as predicted by SwitchSeeker. The two conformations are shown in blue and red, respectively. Left: The schematic representations of the two conformations, as used throughout the article. b, The set-up of mutation–rescue experiments. The switching regions are color coded as in a. A-U and C-G base pairing is shown with compatible shapes (triangle and half-circle). The two conformations of the switch reside in the equilibrium state. Mutation of the Box 3 region disrupts the base pairing between the Box 1 and the Box 3 regions. This causes a shift of the equilibrium towards conformation 2. Rescue mutation of the Box 1 switching region restores the base pairing between Box 1 and Box 3, but at the same time it disrupts the base pairing between Box 1 and Box 2. Therefore, the equilibrium shifts towards conformation 1. c, In vitro SHAPE reactivity of the RORC RNA switch sequence in vitro. Left: SHAPE reactivity profiles for the reference sequence (in gray) and for the mutation–rescue pair of sequences (blue, 65-GT,117-AC; red, 117-AC). Shown is the average for three replicates with the respective error bars (s.d.). The SHAPE reactivity changes in the nonmutated regions are highlighted with bold arrows. Right: Barplots of cumulative SHAPE reactivity in the switching regions. d, Secondary structures of the two conformations of RORC RNA switch predicted by the RNAstructure algorithm⁵⁶ guided by the DMS reactivity data. The base pairing of Box 1 with either Box 3 (conformation 1) or Box 2 (conformation 2) is highlighted by a red frame. The two clusters were identified using the DRACO unsupervised deconvolution algorithm²⁸. e, Accessibility of the Box 2 (x axis) and Box 3 (y axis) regions of the RORC element across cell lines, as measured with DMS-MaPseq (normalized reactivity, see Methods). The cell lines were engineered to express a GFP reporter containing the RORC switch sequence in the 3ʹUTR, and the accessibility of the reporter mRNA was measured with DMS-MaPseq. Linear regression is shown with an orange line. f, Accessibility of the Box 2 (x axis) and Box 3 (y axis) regions of the RORC element in the endogenous RORC mRNA, as measured with DMS-MaPseq (normalized reactivity, see Methods).