RDE-1 (RNAi-DEfective 1) is a primary Argonaute protein required for RNA-mediated interference (RNAi) in Caenorhabditis elegans. The rde-1 gene locus was first characterized in C. elegans mutants resistant to RNAi, and is a member of a highly conserved Piwi gene family that includes plant, Drosophila, and vertebrate homologs.[1]
Upon uptake into the cell, exogenous trigger dsRNA is bound by RDE-4 and cleaved into 21-25nt primary siRNA by a Dicer complex that includes RDE-1.[2] Primary siRNA binding to RDE-1 then promotes the formation of the RNA-induced silencing complex (RISC). Unlike in other Argonautes characterized in Drosophila[3] and humans,[4] the catalytic RNase H motif in RDE-1 has not been shown to exhibit Slicer activity of the target transcript. Instead, RNase H activity in RDE-1 is primarily facilitates siRNA maturation through cleavage of the passenger strand.[5]
The primary Argonaute complex recruits an RNA-dependent RNA polymerase (RdRP) to generate secondary siRNAs, triggering an amplification response. Secondary siRNAs are bound by degenerate secondary Argonautes, which are then directly involved in target transcript degradation.[6] However, RDE-1 shows no stable association with the more abundant secondary siRNAs.[7]
Whereas rde-4 deficiency can be rescued by high concentrations of trigger dsRNA,[8] and secondary Argonaute exhibit functional redundancy,[6] there has been no evidence that RNA-mediated silencing can be reinstated in rde-1 deficient mutants. To this extent, RDE-1 appears to have a qualitatively distinct activity in the exogenous RNAi pathway.[7]
Canonical Argonaute proteins possess three primary domains forming a crescent-shaped base: the PAZ, MID, and PIWI domains. PAZ and MID orient and anchor the double-stranded siRNA by binding to the 3’ and 5’ termini, respectively, leaving the internal nucleotides accessible for base pairing. The PIWI domain folds into an RNase H-like structure, and contains the conserved catalytic triad “DDH” (two aspartate residues, one histidine residue).[9] The crystal structure of RDE-1 has not been formally elucidated, but can be assumed to closely resemble its human homologs.
Mutation of any residues in the RNase H catalytic triad abolishes Slicer activity in human Argonaute protein Ago2, suggesting that the RNase H domain is directly responsible for target mRNA degradation.[10] However, RDE-1 has not been implicated in mRNA-cleavage activity.
Instead, RDE-1 with mutations in the conserved DDH motif exhibit reduced passenger (sense) strand turnover, suggesting that RNase H activity serves to cleave the passenger strand, leaving the guide (antisense) strand accessible for base-pairing to target mRNA.[5] Further, target silencing can be fully restored in DDH motif mutants by loading single-stranded siRNA, suggesting that a downstream component in the RNAi pathway is responsible for Slicer activity.[5] Thus, RDE-1's RNase H domain facilitates siRNA maturation but is not directly involved in cleaving target mRNA transcripts.