Figure 1.

(a) Double-stranded RNA (ds) and single-stranded RNA (ss), both 500 bp long, were incubated with extract from C. elegans embryos. siRNAs are produced in a time-dependent fashion from the dsRNA but not from the ssRNA (time points: 0, 30, and 60 min). (b) siRNA-producing activity can be immunoprecipitated with antisera (I) raised against DCR-1. The preimmune serum (P) is inactive. The left lane contains an RNA marker. (c) siRNA produced by C. elegans extract (C) is ∼2 nt bigger than siRNA produced by Drosophila extract (D). In both reactions a 500-bp dsRNA was used as substrate. (d) Time courses (0, 30, 60 min) with dsRNA of different lengths, using C. elegans extract. siRNA is produced from dsRNA of 35 bp and longer, but the larger dsRNA molecules are clearly better substrates (equal counts and equal mass of RNA were added to each reaction). (e) siRNA production is ATP-dependent. (Lane 1) The substrate (500 bp) was not incubated with extract; (lane 2) a reaction with ATP-depleted extract; (lane 3) ATP is added back to the depleted extract; and (lanes 4,5) nonhydrolyzable ATP analogs are added (ATPγS and ADPNP). (f) Incubation of 500-bp dsRNA with C. elegans embryo extract results in the formation of a ladder, with a spacing characteristic of siRNAs. (Lane 1) Untreated RNA; (lane 2) the same RNA after treatment with extract; (lanes 3,4) identical reactions to lanes 1 and 2, but with a 100-bp dsRNA substrate with a 400-bp single-stranded tail. The laddering stops at the position where the RNA substrate is no longer double stranded.