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 doublestranded 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.

Biochemical and genetic functional dissection of the P38 viral suppressor of RNA silencing

“…2B). The activities of DCL proteins are assisted by dsRNA binding proteins, as exemplified by HYL1 for DCL1, and DRB4 for DCL4 (Dong et al 2008;Fukudome et al 2011). We thus examined the effects of exogenous addition of HYL1 or DRB4 on the dsRNA processing activities in BYL.…”

“…The other three DCLs participate in the production of small interfering RNAs (siRNAs). During post-transcriptional gene silencing (PTGS), DCL4 cooperates with dsRNA binding protein 4 (DRB4) to produce 21-nt siRNA duplexes (Nakazawa et al 2007;Fukudome et al 2011), while DCL2 produces 22-nt siRNA species acting mostly redundantly with the products of DCL4 (Parent et al 2015). DCL3 mediates processing of 24-nt siRNAs that promote RNA-directed DNA methylation (RdDM), potentially resulting in transcriptional gene silencing (TGS) (Blevins et al 2015).…”

Double-stranded RNA binding proteins DRB2 and DRB4 have an antagonistic impact on polymerase IV-dependent siRNA levels in Arabidopsis

“…The DRB4/DCL4 complex is also required to produce siRNAs from exogenous viral dsRNAs. Recently, DRB4 was found to be essential in vitro for DCL4 activity (Qu et al 2008;Fukudome et al 2011). DRB2, DRB3, and DRB5 were reported not to be involved in small RNA biogenesis (Curtin et al 2008), and their functions remained elusive.…”

Biogenesis of <i>trans</i>-acting siRNAs, endogenous secondary siRNAs in plants

“…RDR6-synthesized dsRNAs are processed into 21 nt tasiRNA duplexes with a 2 nt overhang at both 3′ termini by a heterodimer composed of DCL4 and the dsRNAbinding protein DOUBLE-STRANDED RNA BINDING PROTEIN 4 (DRB4) (Hiraguri et al, 2005;Adenot et al, 2006;Nakazawa et al, 2007;Fukudome et al, 2011). The processing of the dsRNAs by DCL4/DRB4 begins from the miRNA cleavage site in a stepwise manner .…”