Figure 4. Methylation of SHC mRNA by NSUN2 enhances SHC translation.
A. Schematic representation depicting the pGL3-derived reporter vectors used for reporter gene assays. B. HeLa cells were transfected with each of the reporter vectors described in Figure 4A together with a pRL-CMV control reporter. Twenty-four hours later, cells were further transfected with with a siRNA targeting NSun2 and cultured for an additional 48 h. Firefly luciferase activity against Renilla luciferase activity was analyzed. Data represent the means ± SD from 3 independent experiments; significance was analyzed by Student's t test (**, p < 0.01). C. In vitro methylated (Met) or unmethylated (Unmet) luc-5′UTR, luc-5′UTRm, luc-CR, luc-CRm, luc-3′UTR, and luc-3′UTRm reporter transcripts were used for in vitro translation assays. Firefly luciferase activity was measured to reflect the translation efficiency. Data represent the means ± SD from 3 independent experiments; significance was analyzed by Student's t test (**, p < 0.01). D. Cells described in Figure 1B were used for isolating the polysomal fractions. RNA prepared from the fractions was subjected to RT-qPCR analysis to assess the presence of SHC mRNA and β-Actin (ACTB) mRNA in the polysomal fraction. Data represent the means ± SD from 3 independent experiments; significance was analyzed by Student's t test (**, p < 0.01).