Reg1 and Reg3 recognize the tandem UAU-SL structures present in the Nfkbiz 3’ UTR. (A) Phosphoimage of SDS-PAGE of radiolabelled Reg3-RNA complexes obtained by immunoprecipitation with anti-FLAG antibody. (B) Pie chart deciphering the distribution of Reg3-binding sites obtained by the intersection of 2 biological HITS-CLIP replicates. (C-D) SL enrichments in Reg3- and Reg1-bound CLIP tags of the 3’ UTR compared with 1000 individual sequence permutations. X-axis indicates hairpin types in which the numbers represent the lengths of stem and loop. ∗P < .01; ∗∗P < .001. (E-F) Sequence logo representations of the recognition motives for Reg3 and Reg1. Stem and hairpin regions are indicated as S and H, respectively, below the x-axis. (G) Schematic representation of the artificial SL structure and its mutant forms (M1 and M2) in which the SL structure is disrupted. (H) Luciferase activity was assessed by using pGL3-βGlo plasmids containing the 3’ UTR with SL, M1, M2, and M1+ M2 as shown in (G). HeLa cells were transfected with pGL3 plasmids together with expression plasmids for WT Reg1, WT, or ND mutant of Reg3 (n = 3 each). (I) Luciferase activity was assessed by using pGL3-βGlo plasmids containing the 3’ UTR with a set of loop sequences. The loop sequences tested are shown in the top of figures. Transfection was performed as in (H) (n = 3 each). (J) Schematic representation of mouse Nfkbiz SL structures targeted by Reg1 and Reg3. M1 and M2 represent mutant sequences that no longer form SL structures. (K) Luciferase activity was assessed by using pGL3-βGlo plasmids containing either WT or mutated SL sequences (M1, M2, or M1+ M2) as shown in (J). HeLa cells were transfected with pGL3 plasmids together with expression plasmids for WT or ND mutant of Reg1 and Reg3 (n = 3 each). Data are representative of 2 to 3 independent experiments (H-I,K). Data are presented as mean ± SD (H-I,K). Statistical significance was calculated by 1-way ANOVA with Holm-Sidak multiple comparisons test (H-I,K).