Specific requirement of DRB4, a dsRNA-binding protein, for the in vitro dsRNA-cleaving activity of Arabidopsis Dicer-like 4

Abstract

Arabidopsis thaliana Dicer-like 4 (DCL4) produces 21-nt small interfering RNAs from both endogenous and exogenous double-stranded RNAs (dsRNAs), and it interacts with DRB4, a dsRNA-binding protein, in vivo and in vitro. However, the role of DRB4 in DCL4 activity remains unclear because the dsRNA-cleaving activity of DCL4 has not been characterized biochemically. In this study, we biochemically characterize DCL4's Dicer activity and establish that DRB4 is required for this activity in vitro. Crude extracts from Arabidopsis seedlings cleave long dsRNAs into 21-nt small RNAs in a DCL4/DRB4-dependent manner. Immunoaffinity-purified DCL4 complexes produce 21-nt small RNAs from long dsRNA, and these complexes have biochemical properties similar to those of known Dicer family proteins. The DCL4 complexes purified from drb4-1 do not cleave dsRNA, and the addition of recombinant DRB4 to drb4-1 complexes specifically recovers the 21-nt small RNA generation. These results reveal that DCL4 requires DRB4 to cleave long dsRNA into 21-nt small RNAs in vitro. Amino acid substitutions in conserved dsRNA-binding domains (dsRBDs) of DRB4 impair three activities: binding to dsRNA, interacting with DCL4, and facilitating DCL4 activity. These observations indicate that the dsRBDs are critical for DRB4 function. Our biochemical approach and observations clearly show that DRB4 is specifically required for DCL4 activity in vitro.

FIGURE 1.

Detection of dsRNA-cleaving activity of DCL4 in Arabidopsis crude extracts. ³²P-labeled 500-bp dsRNAs were incubated with Arabidopsis wild-type (Colombia; Col), dcl3-1, dcl4-2, or drb4-1 crude extract for 2 h at 27°C. The cleavage products were analyzed on 15% denaturing PAGE with 8 M urea. A recombinant human Dicer fragment (hDicer; TaKaRa) was used as a cleavage control to generate size markers for 21-nt small RNA. (White arrowhead) The 500-bp dsRNA substrates; (black arrowhead) 21-nt; (black arrow) 24-nt small RNAs.

FIGURE 2.

Biochemical characterization of in vitro dsRNA-cleaving activity of complexes immunoprecipitated with anti-DRB4 antibody (DRB4 complexes). (A) The dsRNA-cleaving activities of DRB4 complexes purified from WT (Col), dcl4-2, and drb4-1 crude extract by the anti-DRB4 antibody were assayed. The existence of DCL4 in each complex was confirmed by Western blotting with the anti-DCL4 antibody (lower panel). (B) The effects of NaCl concentrations (0–300 mM) on the dsRNA-cleaving activity of DRB4 complexes. (C) DRB4 complexes required 5 mM ATP, 5 mM GTP, and 4 mM Mg²⁺ for the dsRNA-cleaving activity. When both ATP and GTP were present in the reaction mixture (lanes 2,6), 1 mM GTP was used instead. (D) Quantification of 21-nt small RNA products shown in B. Standard condition (lane 3 in B) was set at the arbitrary relative unit “1.0.” (E) Quantification of 21-nt small RNA products shown in C. Standard condition (lane 2 in C) was set at the arbitrary relative unit “1.0.” (White arrowheads) The 500-bp dsRNA substrates; (black arrowheads) the 21-nt small RNA products.

FIGURE 3.

Biochemical characterization of in vitro dsRNA-cleaving activity of complexes immunoprecipitated with anti-DCL4 antibody (DCL4 complexes). (A) The dsRNA-cleaving activities of DCL4 complexes purified from WT (Col), drb4-1, and dcl4-2 crude extracts by the anti-DCL4 antibody were assayed. (B) The effects of NaCl concentrations and the requirement of 5 mM ATP, 5 mM GTP, or 4 mM Mg²⁺ on the dsRNA-cleaving activity of DCL4 complexes. When both ATP and GTP were present in the reaction mixture (lanes 2–7,11), 1 mM GTP was used instead. (C,D) Comparison of the biochemical properties between DRB4 complexes (Fig. 2) and DCL4 complexes. (C) The 21-nt small RNA products shown in B (lanes 2–6) are quantified as described in Figure 2D,E, then plotted on the same graph in Figure 2D. (D) The 21-nt small RNA products shown in B (lanes 7–11) are quantified as described in Figure 2D,E, then plotted on the same graph in Figure 2E. (White arrowheads) The 500-bp dsRNA substrates; (black arrowheads) the 21-nt small RNA products.

FIGURE 4.

Effects of recombinant DRB proteins on the dsRNA-cleaving activity of DCL4 complexes. (A) Purified recombinant GST, DRB1/HYL1, DRB2, DRB3, DRB4, and DRB5 proteins were fractionated by 8% SDS-PAGE, and then stained by CBB. Asterisks indicate the protein of interest. (B–D) Immunoprecipitate (15 μL) purified from drb4-1 by the anti-DCL4 antibody (drb4-DCL4 complexes) was incubated with recombinant proteins for 30 min at 4°C. The dsRNA-cleaving activity of each complex was assayed. (B) The effect of recombinant DRB4 on the dsRNA-cleaving activity of drb4-DCL4 complexes. About 5 to 50 ng of recombinant DRB4 protein was used. (C) The same as B using ∼0.1–312.5 ng of recombinant DRB4 proteins. (D) The effect of each DRB family protein on the dsRNA-cleaving activity of drb4-DCL4 complexes. Twenty-five nanograms of each protein was used. (White arrowheads) The 500-bp dsRNA substrates; (black arrowheads) the 21-nt small RNA products. (Diamond) Short RNA fragments in dsRNA substrates (see Materials and Methods).

FIGURE 5.

Biochemical characterization of mutated DRB4s. (A) Amino acid alignment of dsRBDs. Comparisons among dsRBD1 and dsRBD2 of DRB4, hTRBP2, and Xlrbpa are shown. dsRBD consensus sequences in three conserved regions are shown (Ryter and Schultz 1998). The location of each substitution is indicated by an arrow (histidine 32, H32) and an arrowhead (lysine 133, K133). (B) Gel shift assay showing the dsRNA-binding activity of three kinds of mutated DRB4s (mDRB4; H32A, K133A, and H32A/K133A). Indicated amounts of WT and mutant DRB4 proteins were incubated with ³²P-labeled 100-bp dsRNA for 30 min and analyzed on 6% native PAGE. (C) GST-pull-down assay demonstrating the interaction between sDCL4 and mDRB4s. About 500 ng of GST-fused mDRB4 proteins and the same amount of 6× His-tagged sDCL4 proteins were used as bait and prey, respectively. The interaction was confirmed by Western blotting using the anti-DCL4 antibody. (Arrow) Interacting 6× His-tagged sDCL4. The bands appearing above sDCL4 (an arrowhead) are non-specific (NS) signals. (HK) H32A/K133A. (D) The effect of mDRB4s on the dsRNA-cleaving activity of drb4-DCL4 complexes. About 12.5 ng of recombinant protein was used for each assay. (Black arrowhead) The 21-nt small RNAs (products). (Diamond) Short RNA fragments in dsRNA substrates (see Materials and Methods). (E) Three activities of WT reDRB4 and mDRB4s shown in B–D are summarized. The number of + signs represents the relative strength of each activity, and − signs indicate that the activity was not detected.