Distinct Phases of siRNA Synthesis in an Endogenous RNAi Pathway in C. elegans Soma

Endogenous RNA-directed RNA polymerases (RdRPs) are cellular components capable of synthesizing new complementary RNAs from existing RNA templates. We present evidence for successive engagement of two different RdRPs in an endogenous siRNA-based mechanism targeting specific mRNAs in C. elegans soma. In the initiation stage of this process, a group of mRNA species are chosen as targets for downregulation, leading to accumulation of rare 26 nt 5���-phosphorylated antisense RNAs that depend on the RdRP homolog RRF-3, the Argonaute ERGO-1, DICER, and a series of associated (ERI) factors. This primary process leads to production of a much more abundant class of 22 nt antisense RNAs, dependent on a secondary RdRP (RRF-1) and associating with at least one distinct Argonaute (NRDE-3). The requirement for two RdRP/Argonaute combinations and initiation by a rare class of uniquely structured siRNAs in this pathway illustrate the caution and flexibility used as biological systems exploit the physiological copying of RNA. �� 2010 Elsevier Inc. All rights reserved.We are grateful to Sam Gu, Julia Pak, Leah Frater-Rubsam, Phil Anderson, Chaya Krishna, Cheryl Smith, Ziming Weng, Phil Lacroute, Anton Valouev, and Arend Sidow for their help with experimental protocols and analysis; to Virginia Walbot and Anne Villenueve for comments on the manuscript; to the Caenorhabditis Genetics Center for strains; and for financial support from NIH (R01GM37706 [A.Z.F.], T32HG00044 [J.I.G. and J.M.M.], and R01GM076619 [S.K. and D.M.P.]), Stanford Graduate Fellowship (P.P.), NSF Graduate Fellowship (J.M.M.), and Stanford Dean's Fellowship and Machiah Foundation (A.T.L.)