Abstract

Small RNAs regulate eukaryotic development and immunity. In plants, multiple DICER-LIKE (DCL) proteins produce distinct small RNAs that play diverse functions. These DCL proteins act in a hierarchical manner, with DCL4 outcompeting DCL2 being particularly important for optimal gene expression and plant growth. However, the mechanism of this hierarchical action remains unclear. Here we reveal that the second double-stranded-RNA-binding domain (dsRBD2) of DCL4 interacts with DSRNA BINDING PROTEIN 4 (DRB4), a cofactor essential for DCL4’s function. DRB4 dictates the relative biogenesis of 21- and 22-nucleotide small interfering RNAs derived from TAS loci and coding transcripts. All DCL2 proteins in seed plants lack dsRBD2; however, fusing dsRBD2 to DCL2 enhances its activity, leading to massive production of coding-transcript-derived small interfering RNAs, as well as growth defects and activated stress responses. These findings demonstrate the central role of the dsRBD2–DRB4 module, which enables DCL4 to outcompete DCL2, thereby preventing detrimental gene silencing.