Journal: Nucleic Acids Research

Article Title: 2′- O -Methylation of the second transcribed nucleotide within the mRNA 5′ cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion

doi: 10.1093/nar/gkac722

Figure Lengend Snippet: Methylation of the first transcribed nucleotides in mRNA only partially prevents protein level decrease under IFN-induced stress. Relative protein production levels 72 h after a 5 h IFNα pre-treatment in ( A and B ) A549 and ( C and D ) JAWS II cells. Data were analysed for three independent replicates (each experiment consisted of three technical replicates). Bars for each transcript represent the mean value ± SEM normalized to protein production in mock-treated cells. Statistical significance: * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 (one-way ANOVA with Turkey's multiple comparisons test) (data presenting changes in relative protein production with increasing concentration of IFNα are shown in for A549 and JAWS II cells, respectively). Co-purification of endogenous proteins from lysates of IFNα-treated ( E and F ) A549 (IFNα concentration was 500 U/ml) and ( G and H ) JAWS II (IFNα concentration was 5000 U/ml) cells with biotinylated RNA bearing cap0, cap1, cap2 or cap2-1 with ( E and G ) A or ( F and H ) m 6 A as the first transcribed nucleotide. Human eIF4E, IFIT1 and NCBP1 or murine counterparts (Ifit1b: lower band) were detected in precipitates by western blotting. A 1/15th and 1/21st volume of lysate used for each incubation with beads was loaded as input sample for ( E–G ) and ( H ), respectively; a 1/3rd volume of each eluate was loaded; shorter exposure is presented for input samples than for eluates; *non-specific band.

Article Snippet: The crude mRNAs were purified using NucleoSpin RNA Clean-up XS (740903, Macherey-Nagel). mRNAs were HPLC-purified using an RNASep Prep RNA Purification Column (ADS Biotec) at 55°C, a linear gradient of buffer B (0.1 M triethylammonium acetate pH 7.0 and 50% acetonitrile) from 17.5% to 25.8% in buffer A (0.1 M triethylammonium acetate pH 7.0) over 20 min and a flow rate of 0.9 ml/min. mRNAs from collected fractions were recovered by isopropanol precipitation.

Techniques: Methylation, Concentration Assay, Copurification, Western Blot, Incubation