Specific requirement of DRB4, a dsRNA-binding protein, for the in vitro dsRNA-cleaving activity of Arabidopsis Dicer-like 4 |
| |
Authors: | Fukudome Akihito Kanaya Akihiro Egami Mai Nakazawa Yukihiro Hiraguri Akihiro Moriyama Hiromitsu Fukuhara Toshiyuki |
| |
Institution: | Department of Applied Biological Sciences, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan. |
| |
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. |
| |
Keywords: | DRB4 DCL4 Dicer activity dsRNA-binding protein dsRNA |
本文献已被 PubMed 等数据库收录! |
|