Figure 4

Site-directed mutagenesis of A3G shows requirement for both N- and C–terminal domain zinc dependent deaminase motif residues for site-specific RNA editing.

(a) Chromatograms of cDNAs (single) from ctrl./A3G transfected 293T cells shows the effect of mutations in A3G-NTD (W94A, C97S, W127A) and A3G-CTD (C291S) on C>U RNA editing (edited C is shaded black) in selected genes. (b) Bar graph showing RNA editing level of GOLGA5, KIAA1715 and MED1 in A3G-NTD and -CTD mutant transfectants. RNA editing levels (ratio of edited versus total RNA) are calculated by Sequencher^TM software. The detection limit for relative height of minor peaks was 4.86% (depicted by a dotted line). Mean and SEM (n = 3) (c) Immunoblot showing A3G protein expression in whole cell lysates of 293T cells when transfected with empty vector (control), WT or various A3G-NTD and -CTD mutants. The D128K and P129A mutants were run on separate gels on the same day. The dashed line separates the two gels. Full-length blot is presented in Supplementary Fig. S4 (d) Bar graph depicting RNA editing level of KIAA1715, PRPSAP2, SCD and TM7SF3 cDNAs in WT or mutant transfectants. The detection limit is depicted by a dotted line. Mean and SEM (n = 3). Statistical analysis was performed by one-way ANOVA followed by multiple comparisons of RNA editing levels between WT and the mutants. Editing level is considered 0%, when the Sequencher software detects no secondary T peak. ****=adjusted P value ≤ 0.0001; NS = Not Significant.