.............................................................. A micrococcal nuclease homologue in RNAi effector complexes Amy A. Caudy 1 *, Rene ´ F. Ketting 2 *, Scott M. Hammond 1 †, Ahmet M. Denli 1 , Anja M. P. Bathoorn 1,2 , Bastiaan B. J. Tops 1,2 , Jose M. Silva 1 , Mike M. Myers 1 , Gregory J. Hannon 1 & Ronald H. A. Plasterk 2 1 Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA 2 The Hubrecht Laboratory, Centre for Biomedical Genetics, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands * These authors contributed equally to this work †Present address: Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA ............................................................................................................................................................................. RNA interference (RNAi) regulates gene expression by the cleav- age of messenger RNA, by mRNA degradation and by preventing protein synthesis. These effects are mediated by a ribonucleo- protein complex known as RISC (RNA-induced silencing com- plex) 1 . We have previously identified four Drosophila components (short interfering RNAs 1 , Argonaute 2 (ref. 2), VIG and FXR 3 ) of a RISC enzyme that degrades specific mRNAs in response to a double-stranded-RNA trigger. Here we show that Tudor-SN (tudor staphylococcal nuclease)—a protein containing five staphylococcal/micrococcal nuclease domains and a tudor domain—is a component of the RISC enzyme in Caenor- habditis elegans, Drosophila and mammals. Although Tudor-SN contains non-canonical active-site sequences, we show that puri- fied Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases. Notably, both purified Tudor-SN and RISC are inhibited by a specific competitive inhibitor of micro- coccal nuclease. Tudor-SN is the first RISC subunit to be ident- ified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi. Exposure of cells to double-stranded RNA (dsRNA) can elicit various types of sequence-specific gene silencing 4 . A signature of these silencing events is the involvement of small RNAs of approxi- mately 22–25 nucleotides (nt) that guide the selection of silencing targets 1,5,6 . These short interfering RNAs (siRNAs) or microRNAs (miRNAs) are generated by the processing of silencing triggers by an RNaseIII family nuclease, Dicer 7 . Small RNAs join multicomponent ribonucleoprotein (RNP) complexes, known generically as RISCs, which enforce silencing. Both to address the nature of the RNAi effector machinery in detail, and to examine the relationship between the different effector mechanisms of RNAi, we have biochemically purified a RISC complex from Drosophila that degrades its mRNA target, and have sought to identify its protein and RNA components. In multiple, independent purifications of RISC, we identified, along with previously characterized proteins, a potentially novel com- ponent corresponding to a Drosophila candidate gene, CG7008 (Supplementary Fig. 1). This evolutionarily conserved 103 kDa protein contains five repeats of a staphylococcal/micrococcal nuclease domain (Supplementary Fig. 2). Four of these repeats are intact, whereas the fifth repeat is fused at its amino terminus to a tudor domain, which has been implicated in the binding of modified amino acids 8 . On the basis of this characteristic domain structure, we named the protein Tudor-SN, for tudor staphylococ- cal nuclease. Through each purification step, Tudor-SN co-fractio- nated with known RISC components (Fig. 1a, b and Supplementary Fig. 3). Orthologues of Tudor-SN are found in plants (Arabidopsis 9 ), C. elegans 9,10 , mammals 10,11 and Schizosaccharomyces pombe (A.M.D., unpublished observations), but not in Saccharomyces cerevisiae. To investigate whether a role for Tudor-SNorthologues in RNAi is evolutionarily conserved, we carried out biochemical fractionation of extracts from C. elegans and mammalian cells. We began by preparing cytosolic extracts from synchronized cultures of wild-type C. elegans. As in Drosophila, a large fraction of the miRNA population can be extracted from the ribosomes (Sup- plementary Fig. 4). Size fractionation of extracts derived from adult animals revealed that miRNAs eluted from the column in two peaks, representing ,500 kDa and ,250 kDa complexes (Fig. 1c), similar to what had been observed previously in extracts from Drosophila S2 cells 3 . Three different miRNAs—lin-4, let-7 and mir-52—behaved identically in this assay. By contrast, size fractionation of C. elegans egg extract, and examination of complexes containing mir-40 and mir-52, suggested the presence of only the 500 kDa complex (Fig. 1c). Thus, it seems that miRNAs in C. elegans can inhabit multiple, distinct RNP complexes, and that the partitioning of miRNAs between these complexes may depend on both the identity of the miRNA and the developmental stage of the organism. RISC complexes in C. elegans have not previously been charac- terized. We therefore probed whether Drosophila RISC components co-fractionated with miRNAs in C. elegans extracts. We raised antibodies to F56D12.5 (VIG-1), the worm homologue of Droso- phila VIG, and F10G7.2 (TSN-1), the worm orthologue of Tudor- SN. Both VIG-1 and TSN-1 were enriched in the fractions that contained 250 kDa miRNA (Fig. 1c). By contrast, VIG-1 or TSN-1 did not appear in fractions containing 500 kDa miRNA complex. To test whether putative RISC components are present in Figure 1 Identification and confirmation of Tudor-SN as a component of RISC complexes. a, Drosophila RISC activity was extracted from ribosomes and fractionated on Superose 6. The migration of size markers is indicated. RISC assays and controls for nonspecific activity were carried out as in ref. 1. Western blots were done using antibodies to Ago-2, VIG, FXR and Tudor-SN. b, The active gel-filtration fractions from a were further chromatographed on Source Q. Fractions were analysed as in a. c, Size fractionation of C. elegans ribosome-associated extracts prepared from both eggs and adults. The fractions were analysed by northern blotting. Egg-derived extract is probed for mir-40, the adult extract for let-7. Similar results are obtained when these blots are probed for mir-52. Below, size-fractionated extract from adult animals, analysed by western blotting as indicated for TSN-1 and VIG-1. GFP, green fluorescent protein; Luc, luciferase. letters to nature NATURE | VOL 425 | 25 SEPTEMBER 2003 | www.nature.com/nature 411 © 2003 Nature Publishing Group