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. 2017 Dec 13;46(3):1457–1469. doi: 10.1093/nar/gkx1237

Figure 4.

Figure 4.

A cell-free system for mTOR-regulated translation. (A) Translation of capped and uncapped mRNAs in control and Torin 1-treated extracts. Five nanogram of capped or uncapped mRNA encoding Renilla luciferase was translated in the indicated extracts alone or in the presence of 1 mM cap-analog (7mGDP) and analyzed by luciferase assay. Data are normalized to signal generated by the capped mRNA in the absence 7mGDP (n = 3, error bars are SD). (B) Schematic of TOP and non-TOP reporter mRNA synthesis. mRNAs encoding a 5′ hammerhead ribozyme, Renilla luciferase and a poly-A[60] tail were in vitro transcribed under conditions promoting the cleavage of the ribozyme. TOP or non-TOP RNA 10-mers with or without 7-methylguanosine caps were then ligated to the 5′ ends of the mRNAs using splint ligation. (C) TOP mRNA translation is selectively repressed in extracts from Torin 1-treated cells. A total of 0.5 ng of TOP or non-TOP (nTOP) synthetic mRNAs encoding Renilla luciferase were translated in extracts from cells treated with vehicle (DMSO) or 250 nM Torin 1 for 2 h and analyzed by luciferase assay. Data are normalized to non-TOP levels for each extract. Significance was determined by ANOVA (n = 3, error bars are SD).