Figure 2.

DCL3 preferentially cleaves 37 nt dsRNAs with adenosine or uridine at their 5′ phosphorylated ends. (A) DCL3 cleaves short dsRNAs with adenosine or uridine at their 5′ phosphorylated ends. Four 37 nt dsRNAs, which have a GFP gene sequence containing four different nucleotides at the 5′ phosphorylated end and 2 nt 3′ overhang, were used as substrates for Dicer assays. Cleavage products were analysed by 15% denaturing PAGE. The 24 nt RNA producing activity (DCL3 activity) was calculated as the relative band intensity of 24 nt RNA with respect to the intensity of all bands in each lane following autoradiography, and values are means ± standard deviation for three independent experiments. The schematic figure shows cleavage preference of DCL3. Solid arrows indicate cleavage preference of DCL3. Black stars indicate ³²P. (B) DCL3 efficiently cleaves short dsRNAs with an unstable end. Three 37 nt dsRNAs containing a mismatched base pair at their 5′ phosphorylated ends and one 37 nt perfect dsRNA containing uridine (U) at the 5′ phosphorylated end were used as substrates for Dicer assays. Amounts of cleavage products (24 nt RNA) were calculated as the relative band intensity of 24 nt RNA with respect to the intensity of all bands in each lane following autoradiography. Values in each lane are relative signal intensities of cleavage products, defining the signal intensity in lane U as 1.00. (C) No cleavage preference of DCL3 or DCL4 for 50 nt dsRNAs with various ends as substrates. Four 50 nt dsRNAs with various base pairs at 5′ ³²P-labeled ends (see right figure) and 2 nt 3′ overhangs were used as substrates for Dicer assays. Amounts of cleavage products, 21 nt RNA (DCL4) and 24 nt RNA (DCL3), were calculated as the relative band intensities of 21 and 24 nt RNAs, respectively, with respect to the intensity of all bands in each lane following autoradiography. Values in each lane are relative signal intensities of cleavage products, defining the signal intensity of 24 nt product in lane UA as 1.00.