Translation of a synthetic <i>CAT-FLAG</i> mRNA initiates at two AUG codons in a cap-dependent manner.

2010-11-24T01:52:40Z (GMT) by Daiki Matsuda Vincent P. Mauro
<p><b>A</b>. The <i>CAT-FLAG</i> mRNA used in this study is indicated schematically. It contains four CAA repeats in the 5′ leader and two in-frame AUG codons. FLAG epitopes are indicated by the black bar. This mRNA encodes two proteins with predicted molecular weights of 31 and 28 kDa. <b>B</b>. Western blot analysis. COS-7 cells were transiently cotransfected with plasmid constructs that express the <i>(CAA)<sub>4</sub> CAT-FLAG</i> and the control <i>FLAG-Luc2</i> mRNAs. The wild-type CAT-FLAG construct (WT) contains both AUG codons; AUG1-KO lacks AUG1 (the U has been deleted); and AUG2-KO lacks AUG2 (mutated to AAG). The no promoter construct lacks SV40 promoter/enhancer sequences and the 5′ hairpin construct contains an inverted repeat sequence at the 5′ terminus of the mRNA. A longer film exposure of the blot is shown for the CAT-FLAG protein. <b>C</b>. RT-PCR analysis of <i>(CAA)<sub>4</sub> CAT-FLAG</i> mRNA from COS-7 cells transfected with a plasmid expressing this mRNA (+RT). Control reactions used RNA from untransfected cells (UTC) or did not contain reverse transcriptase (-RT). Size controls for PCR products of unspliced (U) or correctly spliced (S) mRNAs were amplified from plasmids containing an intron (DNA (+) intron) or lacking an intron (DNA (-) intron), using the same PCR conditions in parallel. Two-fold dilutions of the <i>(CAA)<sub>4</sub> CAT-FLAG</i> RNA sample were reverse-transcribed prior to PCR amplification. <b>D</b>. <i>Renilla/Photinus</i> dual luciferase dicistronic analysis. The 5′ leader sequences were tested in the intercistronic region of the dicistronic mRNA. Intercistronic sequences in the parent vector (RP) and the <i>β-globin</i> 5′ leader were used as negative controls for IRES and promoter activities; the EMCV and PV 5′ leaders were used as positive controls for IRES activity. <i>Renilla</i> luciferase (rLuc) activities are indicated by white bars; <i>Photinus</i> luciferase (pLuc) activities are indicated by black bars. Luciferase activities were normalized to 1.0 for activities obtained with the RP construct. Three independent experiments were performed for final quantification; error bars indicate standard deviations.</p>