Fig. 3: YBX3 is a novel RNA m5C binding protein responding to EBV infection.
A Schematic presentation of the functional domains for the YBXs’ family members. CSD, cold shock domain. B Alignment of amino acid sequence of the cold shock domain across YBXs’ family members. Conserved regions are highlighted in red. C Western blot assays showing YBX3 protein levels pulled down by biotin-labeled oligo-C or oligo-m5C RNA probes in HK-1-EBV cells. D Electrophoretic Mobility Shift Assay (EMSA) showing the binding ability of YBX3 cold shock domain (CSD) protein (YBX3CSD) with oligo-C or oligo-m5C RNA probes. E High Performance Liquid Chromatography (HPLC) detecting the RNA m5C level in total (input) or YBX3-bound RNAs (YBX3-IP). YBX3 and LMP1 expression in HK-1 and CNE2 cells before (CTRL) and after EBV infection (EBV + ) as determined by qPCR (F) and western blot analysis (G), respectively. YBX3 and LMP1 expression in HK-1-EBV and CNE2-EBV cells with (LMP1-OE) or without (Vector) LMP1 overexpression as determined by qPCR (H) and western blot analysis (I), respectively. J Western blot analysis evaluating the expression of YBX3 and EMT-related markers in HK-1-EBV and CNE2-EBV cells. Cells were knocked down of YBX3 by shRNA lentivirus and rescued with transient transfection of wild-type YBX3 (WT), mutant lacking m5C modification binding site (MUT), and empty vector (EV) as control. Cells without YBX3 knockdown were scrambled. K Transwell assays assessing the migration abilities of cells described in (J), with statistic of three biological replicates presented on the right. Scale bar, 50 μm. Representative western blot images of three biological replicates with similar results are shown in (C, D, G, I, J). Data in (E, F, H) are Mean ± SD from three independent experiments. Statistical analyses were performed using appropriate tests based on data distribution: two-sided unpaired Student’s t test was applied for (E, F, H), and one-way ANOVA test was used for (K). *P < 0.05, ** P < 0.01, *** P < 0.001.
