FIGURE 3.

(A) ADAR2 inhibited splicing of GRG SS pre-mRNA. In vitro splicing of GRG SS or GRG SS Δ20 pre-mRNA with or without recombinant rADAR2a (A2) was analyzed using RT-PCR at time points as indicated. Products were separated on a nondenaturing 6% polyacrylamide gel. Additional upper bands have been sequenced and correspond to full-length pre-mRNA. The position of precursor and spliced mRNA are indicated on the right, exons are represented by boxes, and introns by lines. Used for size determination was 1 kb PLUS marker (Life Technologies™). The percentage of splicing is indicated below the gel. (B) Splicing and editing are competing in vitro. In vitro editing/splicing reactions with preincubation of either nuclear extract (NE) or recombinant rADAR2a (A2) for 5 min, as indicated. PCR products corresponding to pre-mRNA and mRNA at 30 min of splicing reaction were sequenced, and the chromatograms with the R/G editing site indicated by an arrow are shown. ATP was omitted (-ATP) in the splicing reaction in the middle panel. (C) RNA helicase A (RHA) enhanced in vitro splicing of GRG SS and counteracted splicing inhibition by ADAR2 (A2). In vitro editing/splicing reactions with preincubation of recombinant rADAR2a and/or RHA, as indicated. Two controls are included: a splicing reaction without nuclear extract (No NE), and a control splicing reaction (C) containing an equal volume of ADAR storage buffer without rADAR2a. The positions of precursor and spliced mRNA are indicated to the right of the gel; exons are represented by boxes and introns by lines. Used for size determination was 1 kb PLUS marker (Life Technologies™). The percentage of splicing is indicated below the gel.