Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes "Slicer" activity to RISC, providing the catalytic engine for RNAi.

To equalize X-chromosome dosages between the sexes, the female mammal inactivates one of her two X-chromosomes. X-chromosome inactivation (XCI) is initiated by expression of Xist, a 17-kb noncoding RNA that accumulates on the X in cis. Because interacting factors have not been isolated, the mechanism by which Xist induces silencing remains unknown. Here, we discover a 1.6 kb ncRNA (RepA) within Xist and identify the polycomb complex, PRC2, as its direct target. PRC2 is initially recruited to the X by RepA RNA, with Ezh2 serving as the RNA-binding subunit. The antisense Tsix RNA inhibits this interaction. RepA depletion abolishes full-length Xist induction and H3-K27 trimethylation of the X. Likewise, PRC2 deficiency compromises Xist upregulation. Therefore, RepA/PRC2 is required for the initiation and spread of XCI. We conclude that a ncRNA cofactor recruits polycomb complexes to their target loci.The mouse X-inactivation center harbors several noncoding genes, including Xist (1,2) and its antisense repressor, Tsix (3). On the future Xa (active X), Tsix blocks Xist upregulation and prevents the recruitment of silencing factors in cis. On the future Xi (inactive X), Tsix is downregulated, enabling Xist transactivation and spread of Xist RNA along the chromosome (4). The accumulation of Xist transcripts correlates with a cascade of chromatin changes (5), but how Xist directs these changes is unknown. In principle, the act of transcribing Xist could induce structural changes which could alter chromosome-wide function (1). Alternatively, Xist could work as a transcript (1,2) by recruiting chromatin modifiers or by targeting the X to a specialized compartment (6). Though universally attractive, RNA-based models have remained hypothetical, as Xist-interacting proteins have yet to be identified.To circumvent conventional difficulties with purifying Xist-interacting proteins, we carried out RNA immunoprecipitations (RIP) and asked if Xist RNA can be found in a specific protein complex. We isolated nuclear RNAs and their binding proteins in the native state to avoid fixation artifacts and tested two cell types --mouse embryonic stem (ES) cells, which exist in the pre-XCI state but recapitulate XCI when induced to differentiate; and mouse embryonic fibroblasts (MEFs) which faithfully maintains Xi. Because H3-K27 trimethylation (H3-K27me3) closely follows Xist up-and down-regulation (6-9), we asked if Xist RNA binds the H3-K27 methylase, PRC2, the polycomb complex that includes Eed, Suzl2, RbAp48, and the catalytic subunit, Ezh2 (10). Indeed, α-Ezh2 and α-Suz12 antibodies co-immunoprecipitated Xist RNA (Fig. 1A-D). By contrast, Xist sequences were not detected in α-H3-K27me3, α-H4Ac, and no-antibody controls. Pre-treatment with RNases that digest single-stranded (RNase