Csy4 binding and cleavage are necessary for regulation of transgene expression. (A) Schematics of reporters (left to right): unmodified control, 5′ UTR-HP, ATG-HP, 3′ UTR-HP, and 3′ UTR-HP-Δp(A). Fluorescent images of HEK293 cells expressing each reporter in the absence of Csy4 (B), or presence of either native Csy4 (C), Csy4 H29A (D), or Csy4 R115A/R119A (E). Corresponding transmitted light images are shown as insets in each fluorescent image. (F) Quantitation of GLuc activity at 24 h post-transfection by luminometric analysis for each reporter. Error bars indicate standard deviation of four replicates. Statistical significance was expressed relative to the “No Csy4” control and calculated using a two-tailed Student's t-test ([****] P ≤ 0.0001, [***] P ≤ 0.001).
