RNA secondary structure in the 5′ UTR strongly inhibits translation. (A) Fifty-four nucleotides in the middle of 300NUS2 5′UTR were replaced with a 20 bp-long RNA stem–loop to create 300NUS2 + SL mRNA. Base-pair probabilities determined by the RNAstructure software are shown by color code as indicated. (B,C) Introducing the stem–loop (SL) into the 300NUS2 5′ UTR reduced GFP synthesis. (D–F) A 122 nt-long sequence (122Struct) in the 5′ UTR (gray box in schematics D), which is predicted to fold into multiple stem–loops, inhibits GFP synthesis. (B,E) Mean GFP fluorescence was normalized to that in the GO RNA-ID reporter with a 6 nt-long 5′ UTR. (C,F) Mean GFP/RFP fluorescence ratios normalized to the ratio of GFP and RFP mRNA levels, which were determined by RT-qPCR. GFP/RFP ratio in the GO RNA-ID control was set to 1. Error bars (B,C,E,F) show standard deviations determined from three biological replicates.
