Figure 6. Stability of the upstream RNA secondary structure is inversely correlated with production of protein initiated from the GGC_1274–1276 codon.

(A) Schematic representation of the partial ACAT1 mRNA sequence (nt 1243–1786) and the replacement of AU nucleotides by GC nucleotides in stem-loop I. The detailed sequence of nt 1253–1270 is listed and the replaced nucleotides are underlined. Gray bar, partial ACAT1 mRNA sequence (ACAT1 1243–1786); black bar, 3×Flag coding sequence (3×Flag); filled circle, GGC_1274–1276 initiation codon; hollow circle, AUG_1397–1399 initiation codon.

(B) The expression plasmids depicted in (A) are transiently transfected into AC29, the lysates are prepared and immunoblotting is carried out with anti-ACAT1 antibodies (DM10). Arrows indicate the positions of ACAT1-NT-Flag proteins respectively initiated from GGC_1274–1276 and AUG_1397–1399. The experiments are repeated three times with similar results.

(C) Predicted RNA secondary structures of nt 1253–1270 without or with mutations of stem-loop I depicted in (A). The folding Gibbs free energy (ΔG) of predicted RNA secondary structure is listed beneath each one.

(D) Relative production of ACAT1-NT-Flag protein initiated from the GGC_1274–1276 codon (up panel) and folding ΔG of RNA secondary structures (down panel). The intensity of products from the GGC_1274–1276 codon in (B) is quantified by using the UVP Labwork software (UVP Inc.) for densitometric analysis and normalized to the value of wild type one. The data mean ± SD from three independent experiments. The relative folding ΔG of RNA secondary structures in (C) is shown by using the wild type one as 1.0.