(A) MIWI piRISC, loaded with a synthetic, 5′-32P-radiolabeled, 30-nt piRNA bearing a 2′-O-methyl or hydroxyl 3′ end was incubated in Henmt1em1/em1 testis lysate with 30-nt, fully 2′-O-methylated complementary RNA (cRNA). The mean of the guide abundance is shown (n = 3).
(B) As in (A), a time series is shown for MIWI piRISC incubated in Henmt1em1/em1 testis lysate with 30-nt, fully 2′-O-methylated cRNA.
(C) As in (A), except Henmt1em1/em1 testis lysate was preincubated for 5 min at the indicated temperature before adding MIWI piRISC and cRNA; MIWI piRISC was then incubated in lysate with cRNA for an additional 5 h.