Fig. 4: An RNA-based assay to detect APOBEC3A activity in cells. | Nature Communications

Fig. 4: An RNA-based assay to detect APOBEC3A activity in cells.

From: Quantification of ongoing APOBEC3A activity in tumor cells by monitoring RNA editing at hotspots

Fig. 4

a Schematic representation of the droplet digital PCR (ddPCR) strategy to detect RNA-editing by A3A. b Scatter plots of wild type (green) or edited (blue) DDOST amplified by ddPCR after expression of wild-type A3A or a catalytically inactive mutant A3A (A3AE72A) in U2OS cells. c, d U2OS-derived or RPE1-hTERT-derived cell lines inducibly expressing A3A, A3AE72A, or A3B were induced with doxycycline (DOX) or left uninduced. The level of edited DDOST558C>U and CYFIP13222C>U in U2OS or RPE1-hTERT cells was quantified by ddPCR assay. Error bar: S.D. (n ≥ 3). e HEK-293T cells were transfected with an increasing amount of vector expressing A3A-Flag/GFP. A3A protein levels in HEK-293T cell extracts were analyzed by western Blotting. E.V. empty vector. f Deamination activity on NUP93 and PolyA-TC substrates was measured following incubation with 0.2 μg or 1 μg of HEK-293T cell extracts, respectively, expressing an increasing level of A3A as shown in e. g Quantification of edited DDOST558C>U by ddPCR assay tracked closely with monitoring of cleavage of NUP93 DNA by Electrophoretic Mobility Shift Assay as shown in f. Error bar: S.D. (n = 3). For the ddPCR quantification, RNAs were purified from the same pool of HEK-293T cells shown in e and f. Source data are provided as a Source Data file.

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