Figure 4.

Two-step processing of the 5'-end labelled RNA-I by recombinant DICER protein. (A) In vitro processing of the 5'-end labelled RNA-I by rDICER protein. 5'-end labelled RNA-I RNAs were incubated without rDICER for 120 min (lane 1) and with rDICER for the following time points (0, 10, 20, 30, 40, 50, 60 and 120 min; lane 2-9 respectively). The processing reaction was faster than the results of Figure 3B because the amount of RNA substrate in this reaction mixture was less. The RNA products less than 10 nt look stacked at the end of the gel because of the difficulty in separating efficiently, even at 7.5 M urea denaturing 20% polyacrylamide sequence gel. This experiment was repeated and replicated consistently. M: decade marker. (B) Sequences of band 1 and 2 in Figure 3B identified from Figure 4A and Table 1. Sequences highlighted in gray are 29-nt (band 1) and 23-nt RNA (band 2) from the 5' strand, respectively.