title: Structure of RDE-4 dsRBDs and mutational studies provide insights into dsRNA recognition in the Caenorhabditis elegans RNAi pathway.
abstract: The association of RDE-4 (RNAi defective 4), a protein containing two dsRBDs (dsRNA-binding domains), with long dsRNA and Dcr-1 (Dicer1 homologue) initiates the siRNA pathway in Caenorhabditis elegans. Unlike its homologues in higher eukaryotes, RDE-4 dsRBDs possess weak (micromolar) affinity for short dsRNA. With increasing length of dsRNA, RDE-4 exhibits enhanced affinity due to co-operativity. The linker and dsRBD2 are indispensable for RDE-4's simultaneous interaction with dsRNA and Dcr-1. In the present study, we have determined the solution structures of RDE-4 constructs that contain both dsRBDs and the linker region. In addition to the canonical dsRBD fold, both dsRBDs of RDE-4 show modified structural features such as truncation in the β1-β2 loop that rationalize RDE-4's relatively weak dsRNA affinity. Structure and binding studies demonstrate that dsRBD2 plays a decisive role in the RDE-4-dsRNA interaction; however, in contrast with previous findings, we found ephemeral interaction of RDE-4 dsRBD1 with dsRNA. More importantly, mutations in two tandem lysine residues (Lys217 and Lys218) in dsRBD2 impair RDE-4's dsRNA-binding ability and could obliterate RNAi initiation in C. elegans. Additionally, we postulate a structural basis for the minimal requirement of linker and dsRBD2 for RDE-4's association with dsRNA and Dcr-1.
authors.affiliation: *CSIR-Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500007, India.
authors.forename: Sai Chaitanya
authors.initials: SC
authors.lastname: Chiliveri
authors.affiliation: *CSIR-Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500007, India.
authors.forename: Mandar V
authors.initials: MV
authors.lastname: Deshmukh
mesh_terms: D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D034622:RNA Interference
chemical_list: D017931:DNA Primers; D004247:DNA