The mechanism of DRB7.2:DRB4 mediated sequestering of endogenous inverted-repeat dsRNA precursors in plants

Noncoding transcribable inverted repeat sequences are vital in genome stability, regulation of transposable elements, mutations, and diseases in eukaryotes. In vascular plants, dsRNA Binding Proteins (dsRBPs), DRB7.2 and DRB4, inhibit Dicer-like-protein 3 (DCL3) to stall endogenous inverted-repeat dsRNA (endo-IR dsRNA) mediated gene silencing. As dsRBPs generally assist Dicers, the inhibition of DCL3 by a dsRBP complex is quite enigmatic. Here, we explore how the DRB7.2:DRB4 complex sequesters substrate dsRNA of DCL3 using a structure-based mechanistic approach. Intriguingly, the crucial step of endo-IR dsRNA precursor sequestration is the high affinity complex formation of interacting domains of DRB7.2 (DRB7.2M) and DRB4 (DRB4D3). Next, we establish that DRB7.2 simultaneously interacts with DRB4 and endo-IR dsRNA precursors, where DRB4 contributes towards enhancement in the affinity of the complex with dsRNA, thereby impairing DCL3 mediated cleavage of endo-IR dsRNA precursors. The uniqueness of the DRB4D3 structure implies that the trans-acting (tasi)/siRNA initiation complex formed by DCL4:DRB4 in plants is diverse from its non-plant higher eukaryotes. Overall, we present considerable insights into endo-IR dsRNA precursors regulation in plants and indicate a differential evolution of RNAi initiation complexes between plants and other higher eukaryotes.