Figure 8
From: Capping Enzyme mRNA-cap/RNGTT Regulates Hedgehog Pathway Activity by Antagonizing Protein Kinase A
A conserved function of human mRNA-cap in Hh pathway regulation. (A–A”) A wing disc expressing HA-h-RNGTT and mRNA-cap RNAi with MS1096 was immunostained for Ci, ptc-lacZ and Smo. Defects in Ci, ptc-lacZ and Smo level were rescued by HA-h-RNGTT overexpression. (A”’,B”’,C”’,D”’) Adult wings expressing HA-h-RNGTT (A”’), HA-h-RNGTT MP (B”’), HA-h-RNGTT MG (C”’) or HA-h-RNGTT K294A (D”’) in mRNA-cap knockdown background. Only expression of HA-h-RNGTT could rescue the adult wing defect induced by mRNA-cap RNAi. (B–D”) Wing discs expressing mRNA-cap RNAi in conjunction with HA-h-RNGTT MP (B–B”) or HA-h-RNGTT MG (C–C”) or HA-h-RNGTT K294A (D–D”) were immunostained for Ci, ptc-lacZ and Smo. The defects in Ci, ptc-lacZ and Smo levels were not rescued by the expression of HA-h-RNGTT MP, HA-h-RNGTT MG or HA-h-RNGTT K294A. (E–H) Gli-luciferase (Gli-luc) reporter assays in NIH/3T3 cells transfected with the indicated constructs in the absence (E,F) or presence (G,H) of Shh treatment. Gli luciferase activities were normalized to Renilla luciferase activities. RNGTT increased whereas RNGTTK294A or knockdown of RNGTT decreased Shh pathway activity. Co-transfection with PRKAR1A restored the pathway activity. The knockdown efficiency of RNGTT was assessed through PCR. Actin acts as a loading control. All images are representative of three independent experiments. Data presented are the average of three independent experiments and error bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001, t-test.
