Figure 3

Processing of the hairpin RNA with 5' overhangs by recombinant DICER protein. (A) Hairpin RNA with 5' overhangs (RNA-I) and probes used in this study. RNA-I was a hairpin RNA with 5' overhangs based on the "pre-mir-21 RNA" sequence. The secondary structure was predicted using the CentroidFold program [49]. The solid line shows the position of probe-1, the dashed line shows the position of probe-2 and the dotted line shows the position of probe-3. (B-D) Time-course analysis of the processing of RNA-I by the rDICER protein. RNA-I RNAs were incubated with rDICER in vitro for the indicated time points (0, 10, 20, 30, 40, 50, 60, 75, 90, 105 and 120 min). The RNAs processed by rDICER were detected using probe-1, probe-2 and probe-3 (B-D, respectively) by Northern blotting. The gray arrow shows the band of unprocessed RNA-I and the black arrow shows the bands of small RNA processed from the 5' strand, 3' strand and loop region of RNA-I respectively. M: decade marker. (E) The processing of RNA-I by the rDICER protein at a longer incubation time. RNA-I RNAs were incubated with rDICER for 30 min and 16 hours. The RNAs processed from the 5' strand of RNA-I were detected using probe-1 by Northern blotting.