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. Author manuscript; available in PMC: 2011 May 3.
Published in final edited form as: Cell Res. 2008 Sep;18(9):921–936. doi: 10.1038/cr.2008.66

Figure 2. Translation initiation from GGC1274–1276 codon is mediated by IRES.

Figure 2

(A) Schematic representation of the partial ACAT1 mRNA sequences (nt 1243–1786) and its truncated form (nt 1397–1786) without or with a 5'-stable hairpin. The stable hairpin (ΔG = −57 kcal/mol) is located at 5'-end of the partial ACAT1 mRNA sequences and its truncated form in the plasmids phNTF and phNTF-D5. The vicinity of the GGC1274–1276 codon was deleted (Δ1243–1396) in the negative control plasmids pNTF-D5 and phNTF-D5. Gray bar, ACAT1 mRNA sequence (ACAT1 1243–1786); black bar, 3×Flag coding sequence (3×Flag); filled circle, GGC1274–1276 initiation codon; hollow circle, AUG1397–1399 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 GGC1274–1276 and AUG1397–1399. The experiments are repeated three times with similar results.

(C) Schematic representation of the two cistrons without or with the stable hairpin. The first and second cistrons are the whole AUG-ORFs of Renilla luciferase and Firefly luciferase in the plasmids pRnF, phRnF and pRhnF as the negative controls. The expression plasmids pRAF, phRAF and pRhAF contain the second cistrons by fusing the vicinity of GGC1274–1276 codon (nt 1243–1396) with the 5'-end of whole AUG-ORF of Firefly luciferase. The stable hairpin depicted in (A) is located at 5'- or 3'-end of the first cistron. Gray bar, the vicinity of GGC1274–1276 codon (ACAT1 1243–1396); hatched bar, whole AUG-ORF of Renilla luciferase (Rluc); white bar, whole AUG-ORF of Firefly luciferase (Fluc); others representing the same in (A).

(D) The expression plasmids depicted in (C) are transiently transfected into AC29, the lysates are prepared and immunoblotting is carried out with anti-Fluc antibody and anti-Rluc antibody respectively. The immunoblotting result with anti-Fluc antibody is shown on the top panel and arrows indicate the positions of the fused ACAT1-Fluc protein initiated from the GGC1274–1276 and Fluc protein initiated from AUG1397–1399. The immunoblotting result with anti-Rluc antibody is shown on the bottom panel and an arrow indicates the position of Rluc protein. The experiments are repeated three times with similar results.