The fractionation of molecular weight standards (MW) is indicated. in a broad range of organisms1. InCaenorhabditis elegans, exogenous (exo) RNAi is triggered by foreign dsRNA, which is recognized by the type III ribonuclease DCR-1 and its co-factors the dsRNA-binding domain protein RDE-4 and the putative RNA helicase DRH-1 (ref.2). DCR-1 processes the dsRNA into short-interfering (si) RNAs, which are specifically loaded onto the Argonaute RDE-1 (ref.3,4) and serve as a guide, directing RDE-1 to a target mRNA through base-pair interactions. Recognition of a target by RDE-1 activates a siRNA amplification system that is dependent on the cellular RNA-dependent RNA polymerases (RdRPs) RRF-1 and EGO-1 (ref.47). These so-called secondary siRNAs are synthesizedde novoby RdRPs and selectively loaded onto a family of worm-specific Argonautes (WAGOs)4, which effect silencing by transcriptional8and-or Anemarsaponin E post-transcriptional means9. Endogenous (endo) RNAi pathways refer to mechanisms whereby siRNAs are derived from, and converge back on, endogenous loci. Endo-RNAi pathways are pervasive and remarkably diverse10, but many of the underlying mechanistic details are unknown. InC. elegans, endo-RNAi pathways are required for a variety of cellular processes, including the assembly and-or maintenance of chromosome integrity11,12,13, transposon silencing and transcript surveillance14, and a gene-specific regulatory mechanism during early larval development15. Interestingly, many of these endo-RNAi pathways appear to function independently of DCR-1 (ref.14). The ERI endo-RNAi pathway is a DCR-1-dependent pathway that functions during sperm development as well as during embryogenesis13,16,17. In this pathway, DCR-1 is required for the biogenesis of endo-siRNAs called 26-G-RNAs, which are 26 Anemarsaponin E nucleotides NPM1 (nt) with a bias for a monophosphorylated 5-guanosine (G)18,19. The 26-G-RNAs are loaded into specific Argonaute family members ALG-3 and ALG-4 in developing sperm cells16and ERGO-1 in the early embryo20, respectively. In the embryo, the ERI endo-RNAi mechanism activates and competes for the same RdRP-dependent siRNA amplification and WAGO system that is required for the exo-RNAi pathway13,20,21. The molecular events that initiate ERI endo-RNAi are unknown. Our previous work identified a number of DCR-1-associated proteins that are required for ERI endo-RNAi, including the RdRP RRF-3, the Dicer-related helicase DRH-3, the Tandem-tudor domain protein ERI-5, the SAP-exonuclease ERI-1b, as well as the novel proteins ERI-3 and ERI-9 (ref.13,17,21,22). Recent studies have also revealed that ERI endo-RNAi is dependent on the N-terminal helicase domain of DCR-1 (ref.22). In this study, we sought to refine the functional architecture of the exo- and endo-RNAi machineries inC. elegans. We demonstrate that two separate DCR-1 complexes are assembled to initiate the exo- and the ERI endo-RNAi pathways. We identify two distinct subunits within the ERI complex and show that an RdRP module composed of RRF-3, DRH-3 and ERI-5 is tethered to DCR-1 to potentiate ERI endo-RNAi. In addition to solving the molecular phenotype oferi-5, we demonstrate that paralogous but precisely specified RdRP modules govern DCR-1-dependent and -independent RNAi pathways. == Results == == DCR-1 is in distinct exo- and endo-RNAi complexes == DCR-1 associates with proteins involved in microRNA-mediated silencing, exo-RNAi and endo-RNAi pathways13. To examine the distribution of DCR-1 complexes in the exo- and endo-RNAi pathways, we used gel-filtration chromatography to resolve DCR-1 complexes in an embryo extract (Fig. 1a). Western blot analyses revealed that DCR-1 was present in fractions ranging 600950kDa in molecular Anemarsaponin E weight (MW) that could be separated into two populations. The peak fractions of DCR-1 (600700kDa) co-fractionated with factors required for exo-RNAi, including the putative RNA Anemarsaponin E helicase DRH-1 and two isoforms of the dsRNA-binding protein RDE-4 (43 and 46kDa). A second complex population, representing roughly 43%.