ABSTRACT
DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. The enzyme has two tandem double stranded RNA binding domains (dsRBDs) in its C-terminus. Here we show that the first of these domains binds precursor RNA fragments when isolated and cooperates with the second domain in the recognition of substrate RNA. Remarkably, despite showing RNA binding activity, this domain is intrinsically disordered. We found that it acquires a folded conformation when bound to its substrate, being the first report of a complete dsRBD folding upon binding. The free unfolded form shows tendency to adopt folded conformations, and goes through an unfolded bound state prior to the folding event. The significance of these results is discussed by comparison with the behavior of other dsRBDs.
Subject(s)
Arabidopsis Proteins/chemistry , Cell Cycle Proteins/chemistry , RNA-Binding Proteins/chemistry , Ribonuclease III/chemistry , Arabidopsis Proteins/metabolism , Cell Cycle Proteins/metabolism , Intrinsically Disordered Proteins/chemistry , Models, Molecular , Protein Binding , Protein Stability , Protein Structure, Tertiary , RNA/chemistry , RNA/metabolism , RNA Folding , RNA-Binding Proteins/metabolism , Ribonuclease III/metabolismABSTRACT
HYL1 is a double-stranded RNA binding protein involved in microRNA processing in plants. HYL1 enhances the efficiency and precision of the RNase III protein DCL1 and participates in microRNA strand selection. In this work, we dissect the contributions of the domains of HYL1 to the binding of RNA targets. We found that the first domain is the main contributor to RNA binding. Mapping of the interaction regions by nuclear magnetic resonance on the structure of HYL1 RNA-binding domains showed that the difference in binding capabilities can be traced to sequence divergence in ß2-ß3 loop. The possible role of each domain is discussed in light of previous experimental data.