Hypoxia induces a functionally significant and translationally efficient neuronal NO synthase mRNA variant
J. Clin. Invest. Michael E. Ward, et al. 115:3128 doi:10.1172/JCI20806 [Go to this article.]

Figure 8
Effect of hypoxia on translation of nNOS leader exons. (A) Design of the monocistronic luciferase reporter constructs. Varied human nNOS 5′-UTR sequences were placed upstream of the luciferase ORF. (B) Effect of the varied nNOS leader sequences on the translational efficiency of reporter RNAs. In vitro–transcribed, capped, nNOS 5′-UTR/luciferase reporter RNAs were transiently translated in rabbit reticulocyte lysates or transiently transfected into cultured C2C12 myocytes. Data are expressed as the mean ± SEM of 3 independent experiments, each performed in triplicate. *P < 0.05 for difference from Ex2-Long (Ex2-L) value. Ex2-S, Ex2-Short; Vct, vector only. (C) Studies assessed the effect of normoxia (white bars) versus hypoxia (5 hours; black bars) on reporter Firefly luciferase activity in cultured myocytes (C2C12 cells) transiently transfected with RNA constructs containing the nNOS Ex-1c, Ex2-Short, and Ex2-Long leader sequences. Shown is the mean ± SEM of 3 independent experiments, each performed in triplicate. (D) Design of the bicistronic luciferase reporter constructs. Varied human nNOS 5′-UTR sequences were inserted into the multiple cloning site (MCS) between the Renilla and Firefly luciferase ORFs. pRhrvF and pRpolioF containing the human rhinovirus IRES element and poliovirus IRES element, respectively, served as positive controls for IRES activity. (E) Ratio of Firefly to Renilla luciferase activity (FL/RL) relative to the activity of the control construct lacking any nNOS 5′-UTR sequences (pRF) under normoxic (white bars) and hypoxic (5 hours; black bars) conditions. Studies assessed reporter activity in cultured myocytes (C2C12 cells) transiently transfected with bicistronic RNA constructs. Data are expressed as the mean ± SEM of 3 independent experiments, each performed in triplicate.