Journal: RNA

Article Title: IGF2BP1 enhances HCV IRES-mediated translation initiation via the 3′UTR

doi: 10.1261/rna.1578409

Figure Lengend Snippet: HCV IRES-mediated translation initiation is enhanced by the HCV 3′UTR in vitro. (A,B, upper panel) Schematic representation of HCV 5′UTR and HCV 3′UTR bearing Luc reporter mRNAs. (Lower panel) Cytoplasmic Huh7 cell extract was used in cell-free translation reactions programmed with the [32P] trace-labeled mRNAs HCV Luc or HCVΔ3′UTR Luc for the times indicated. Luc activity is expressed as Luc units. Error bars denote the standard deviation from the mean of three different experiments. [32P] trace-labeled mRNAs were extracted from the translation reaction at the time points indicated. [32P] trace-labeled 5′cap sORF mRNA was used as an extraction control. Extracted mRNAs were analyzed by agarose gel electrophoresis. The incorporation of [32P] was determined by phosphorimaging (Storm 860 Phosphor Imager; Molecular Dynamics, Image Quant software) and expressed as a percentage of signal intensity. Error bars denote the standard deviation from the mean of three different experiments. (C) [32P]-labeled HCV sORF mRNA (left panel) or HCVΔ3′UTR sORF mRNA (right panel) was incubated in Huh7 cell extract in the presence of 2 mM cycloheximide for 1 and 15 min, as indicated. (D) [32P]-labeled HCV sORF mRNA (left panel) or HCVΔ3′UTR sORF mRNA (right panel) was incubated in Huh 7 cell extract in the presence of 2 mM cycloheximide and 5 mM GMP-PNP for 1 and 15 min, as indicated. (C,D) Translation initiation complexes were allowed to assemble for the time indicated and subsequently resolved by centrifugation on 5%–25% linear sucrose gradients. After fractionation from the bottom to the top of the gradient, the radioactivity was monitored, expressed as a percentage of incorporation, and plotted against the fraction number. RNA extracted from 22 fractions was analyzed on agarose gels and visualized by ethidium bromide staining. The positions of 18S and 28S rRNA are indicated. The distribution of rpS3 and eIF6 in the sucrose gradient fractions, determined by Western blotting, indicates the position of 80S ribosomes and ribosomal 60S and 40S subunits, respectively.

Article Snippet: The incorporation of [ 32 P] was determined by phosphorimaging (Storm 860 PhosphorImager; Molecular Dynamics Image Quant software) and expressed as a percentage of signal intensity.

Techniques: In Vitro, Labeling, Activity Assay, Standard Deviation, Extraction, Control, Agarose Gel Electrophoresis, Software, Incubation, Centrifugation, Fractionation, Radioactivity, Staining, Western Blot

Journal: RNA

Article Title: IGF2BP1 enhances HCV IRES-mediated translation initiation via the 3′UTR

doi: 10.1261/rna.1578409

Figure Lengend Snippet: IGF2BP1 enhances HCV IRES-mediated translation via the 3′UTR in rat primary hepatocyte extract. (A) Detection of IGF2BP1 and vinculin (control) in Western blot assays: (lane 1) recombinant IGF2BP1; (lane 2) cytoplasmic Huh 7 cell extract; (lane 3) cytoplasmic rat primary hepatocyte extract; (lane 4) total rat primary hepatocyte extract; and (lane 5) total extract of rat testis (antibody control). (B) Cytoplasmic extract of rat primary hepatocytes was used in cell-free translation reactions, programmed with [32P] trace-labeled reporter mRNAs HCV Luc or HCVΔ3′UTR Luc for the times indicated. Luciferase activity is expressed as relative Luc units. Error bars denote the standard deviation from the mean of three different experiments. (C) [32P] trace-labeled mRNAs were extracted from the translation reaction at the time points indicated. [32P] trace-labeled 5′cap sORF mRNA was used as an extraction control. Extracted mRNAs were analyzed by agarose gel electrophoresis. The incorporation of [32P] was determined by phosphorimaging (Storm 860 PhosphorImager; Molecular Dynamics Image Quant software) and expressed as a percentage of signal intensity. Error bars denote the standard deviation from the mean of three different experiments. (D) Reporter mRNAs HCV Luc or HCVΔ3′UTR Luc were incubated in cytoplasmic extracts of rat primary hepatocytes in the presence of increasing amounts of recombinant IGF2BP1. Luciferase activity is expressed as relative Luc units. Error bars denote the standard deviation from the mean of three different experiments. Translation was set to 100% in the absence of recombinant IGF2BP1. In the presence of recombinant protein, data were analyzed with the t-test (two-tailed), (***) p < 0.0005.

Article Snippet: The incorporation of [ 32 P] was determined by phosphorimaging (Storm 860 PhosphorImager; Molecular Dynamics Image Quant software) and expressed as a percentage of signal intensity.

Techniques: Control, Western Blot, Recombinant, Labeling, Luciferase, Activity Assay, Standard Deviation, Extraction, Agarose Gel Electrophoresis, Software, Incubation, Two Tailed Test