Figure 2

Processing of the precursor miRNA by recombinant DICER protein. (A) Precursor miRNA (pre-mir-21 RNA) and probe used in this study. The 5' end nucleotide of pre-mir-21 RNA was modified (U to G) from human pre-hsa-mir-21 RNA sequence registered in miRBase 14.0 [46–48] in order to facilitate the in vitro transcription reaction. The secondary structure was predicted using the CentroidFold program [49]. The solid line shows the position of probe-1. (B) Detection of the rDICER-processed products by Northern blotting. Pre-mir-21 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 by Northern blotting. The gray arrow shows the band of pre-mir-21 RNA and the black arrow shows the band of miRNA processed from the 5' strand of pre-mir-21 RNA. The asterisk shows the nicked product (~37 nt) similar to a previous report [29].