FIGURE 3.

KREPA6 repression results in a substantial reduction of RNA editing activities in vitro. Crude mitochondrial extracts of RNAi-A6 cells induced with tet for the indicated number of days were fractionated on glycerol gradients. Odd-numbered gradient fractions were assayed for (A) pre-cleaved insertion and (B) pre-cleaved deletion editing activities in vitro. The radiolabeled input RNAs, input RNA with two Us added (+2U), or four Us removed (−4U), ligated products of unprocessed 5′ and 3′ input RNAs (ligated), and edited products are indicated. Positive control reactions (+) were performed using a fraction of ∼20S editosomes that contains peak editing activity. The region corresponding to 20S editosomes is indicated, based on Western blot analyses of the gradients with antibodies specific for known editosome proteins as in Figure 2 (data not shown).