Figure 6
G-quadruplex and uAUGs played a synergistic role in the inhibitory effect of NRXN2α-5′UTR. SH-SY5Y cells (A–C), SK-N-SH and U-87MG cell (D,E) were co-transfected with various luciferase reporter plasmids and p3PRluc. Firefly luciferase activity was measured 24–48 h after transfection, and Renilla luciferase activity was used to normalize for transfection efficiency. Data are shown as relative values over the samples transfected with plasmids containing wild type sequences of NRXN2α-5′UTR or control plasmids. (A) Comparison of luciferase activity between reporter with wild type and G4M10 mutant of full-length 5′UTR. (B) Comparison of luciferase activity among reporter with wild type and corresponding mutants of 5′UTR-401/462 nt. (C) Comparison of luciferase activity among reporter with wild type, uAUG triple mutation only and combined mutations of uAUGs and G4 sequence in full-length 5′UTR. (D) Comparison of luciferase activity between reporter with wild type of full-length 5′UTR and control plasmids in SK-N-SH and U-87MG cell. (E) Comparison of luciferase activity among reporter with wild type, mutant with triple mutations of three uAUGs and mutant of both three uAUGs mutation and G-quadruplex mutation in full-length 5′UTR. (F) Western Blot of luciferase protein. U-87MG cells were transiently co-transfected with luciferase reporters and EGFP expression plasmid. All values above were expressed as means ± S.D., N = 3. ***P < 0.001, **P < 0.01 and *P < 0.05 by One-Way ANOVA (B,C,E) or t-test (A,D). The full-length blots for (F) are presented in Supplementary Fig. 2.
