Journal: Nucleic Acids Research
Article Title: Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity
Figure Lengend Snippet: Contrary to what occurs in β-globin mRNA, α-globin mRNAs allow for efficient translation re-initiation, but only partially contributes to NMD inhibition. ( A ) Schematic representation of the studied human α-globin mRNAs. Vertical lines represent translation initiation (AUG) or termination [native (UAA) or premature (PTC)] codons. The conversion of the two potential re-initiation AUGs, at codons 32 and 76, to ACG codons is indicated by short and thick vertical lines. The name of each transcript is specified on the right. ( B ) A representative RPA of RNA isolated from HeLa cells transiently transfected with each gene construct is shown. Identification of the α-globin and the puromycin-resistance (puro R ) protected fragments is indicated to the right of the autoradiograph. Levels of α-globin mRNA were quantified relatively to the puro R mRNA, and these values are plotted above each respective lane (average and standard deviations) normalized to the expression level of the αWT gene. A 2-fold RNA sample (2x αWT) from HeLa cells transfected with a αWT gene was also analyzed to demonstrate that the experimental RPA was carried out in probe excess. For each case, three independent experiments were performed. P -values were determined using a student's t -test and refer to the comparison with the corresponding original nonsense-mutated transcript levels, after normalization to the wild-type control. ( C ) The 5′-part of α-globin gene upstream of codon 32 (AUG), or upstream of codon 55 (AUG) of β-globin gene, was fused with the firefly luciferase open reading frame (ORF). Volume-shaped rectangles represent luciferase ORFs, dark-gray rectangles represent α-globin exons and light-gray rectangles represent β-globin exons. The initiation or potential re-initiation, as well as termination [native (UAA) or premature (PTC)] codons, are represented as black vertical lines. A positive control construct was created for each hybrid gene, in which the translation initiation codon for α- or β-globin gene was mutated to AUA. HeLa cells were transiently cotransfected with firefly and Renilla luciferase-containing plasmids. Luciferase activity was analyzed 16 h after transfection. In parallel, both firefly and Renilla luciferase mRNA levels were determined by semi-quantitative RT-PCR. The activities of firefly and Renilla luciferase were calculated relative to their respective mRNA levels (Relative mRNA Levels, left bars), and then the resulting values for firefly luciferase activity/mRNA were quantified relative to those of Renilla luciferase activity/mRNA. The obtained luciferase relative activities from hybrid mRNAs were normalized to the relative activity of the uncoupled firefly luciferase (Luc). The average values and standard deviations from three independent experiments corresponding to three independent sets of transfections are shown.
Article Snippet: The βWT-pk, β23-pk and β39-pk gene variants were constructed by replacing the first 19 codons of a native β-globin ORF by a 19-codon sequence resulting in a pseudoknot structure in the mRNA , using the ExSite PCR-Based Mutagenesis Kit (Stratagene) as indicated by the manufacturer, with mutagenic primers #83 and #84, and the βWT and β39 genes as DNA templates, or primers #83 and #85 using the β23 gene as DNA template.
Techniques: Inhibition, Recombinase Polymerase Amplification, Isolation, Transfection, Construct, Autoradiography, Expressing, Luciferase, Positive Control, Activity Assay, Quantitative RT-PCR