Figure 5

BRAT represses expression of reporter transcripts in S2 cells. (A) Schematic of luciferase reporters containing the Firefly luciferase ORF fused to a 3’UTR carrying either six wild-type (luc-wt) or six mutated (luc-mut) BRAT-binding sites. (B) Luciferase activity from each of the reporters was measured after transfection into S2 cells, and normalized to a Renilla luciferase control. Luciferase activity was reduced to approximately 40% by the wild-type sites compared to the mutated sites. (C) Normalized luciferase activity (Firefly/Renilla) from the luc-wt reporter relative to the luc-mut reporter was measured upon treatment with either control dsRNA or one of two dsRNAs directed against brat (Additional file 7 quantifies BRAT knockdown). RNAi-mediated knockdown of brat abrogated repression of the luc-wt reporter, resulting in an approximately three-fold increase in expression of the luc-wt reporter relative to the mutated reporter. (D) Normalized luciferase mRNA levels (Firefly/Renilla) from the luc-wt reporter relative to the luc-mut reporter were measured by RT-qPCR upon treatment with control dsRNA or either of the two dsRNAs directed against brat. The steady state level of the luc-wt reporter was approximately 75% that of the mutated reporter, indicating that BRAT-binding sites reduce steady-state RNA levels. RNAi-mediated knockdown of brat abrogated this repression, resulting in an approximately two-fold increase in expression of the wild-type reporter relative to the mutated reporter. Comparing the results in (C) and (D), both the repression of the wild-type reporter relative to the mutated reporter and the increase in expression of the wild-type reporter upon BRAT knockdown, were less for steady state mRNA levels (D) than for luciferase activity (C), indicating that BRAT regulates both mRNA stability and translational repression. Values represent average of three biological replicates (B and C) or two biological replicates (D) and error bars indicate standard deviation. Student’s t-test P values are indicated.