Figure 5. Pum binds to the potential NRE-like sequence of EGFR signaling components.

(A) (Upper) Schematic drawing of the yeast three-hybrid assays. An RNA containing the NRE and MS2 sequence recruits both Gal4 transcriptional activation domain (GAD)-MS2 coat protein (CP) fusion protein (GAD-MS2 CP) and lexA DNA binding domain (DBD)-Puf fusion protein (LexA-Puf). The resultant ternary complex leads to the expression of lacZ coding sequence through binding to lexA binding sequence (LexAop). (Lower) Yeast YPH500 cells harboring the lexA_op-LacZ reporter, LexA DBD-Puf and GAD-MS2 CP were transformed with vectors that allow for expression of diverse NRE-MS2 transcripts as indicated. Liquid β-galactosidase assays were carried out for transformants. The mean ± SD values were obtained from at least three independent experiments and are presented on the Y-axis. (B) (Upper) Schematic diagram of a reporter containing luciferase (luc) coding sequence with CMV promoter (black arrow) and NREs (black box) in its 5′- and 3′-UTR, respectively. (Lower) HEK293 cells were transfected with the luciferase reporter plasmid containing NRE-like sequence as indicated, alone (−) or in combination with pum expression vector (+). Luciferase activities were measured and the mean ± SD values obtained from at least three independent experiments performed in triplicate (*, P < 0.05). The P-values were obtained by student’s t-test in SigmaPlot.