Abstract
In RNA silencing pathway which is widely conserved among eukaryotes, the specific interaction between a Dicer-family protein and a dsRNA binding protein plays an important role. In this study, we biochemically analyzed the function of Arabidopsis thaliana dsRNA binding protein 4 (DRB4) in the dsRNA cleavage activity of Dicer-like 4 (DCL4). We prepared crude extracts from Arabidopsis seedlings, and incubated them with 500bp dsRNA derived from actin gene. As a result, the extracts from wild-type plants produced 21nt small RNA while the extracts from dcl4-2 and drb4-1 mutants did not. Then, we purified the DRB4 complexes from crude extracts by co-immunoprecipitation using anti-DRB4 antibody. The DRB4 complexes produced 21nt small RNA from 500bp dsRNA in vitro. DCL4 complexes purified by anti-DCL4 antibody showed cleavage activity similar to the DRB4 complexes. The DCL4 complexes from the drb4-1 extracts could not produce any small RNAs, but the addition of recombinant DRB4 to these complexes recovered the activity of 21nt small RNA production. These results suggest that DRB4 plays an essential role in the DCL4-dependent dsRNA cleavage and production of 21nt small RNA.
