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.

Small RNAs associate with Argonaute proteins and serve as sequence-specific guides to regulate messenger RNA stability, protein synthesis, chromatin organization and genome structure. In animals, Argonaute proteins segregate into two subfamilies. The Argonaute subfamily acts in RNA interference and in microRNA-mediated gene regulation using 21-22-nucleotide RNAs as guides. The Piwi subfamily is involved in germline-specific events such as germline stem cell maintenance and meiosis. However, neither the biochemical function of Piwi proteins nor the nature of their small RNA guides is known. Here we show that MIWI, a murine Piwi protein, binds a previously uncharacterized class of approximately 29-30-nucleotide RNAs that are highly abundant in testes. We have therefore named these Piwi-interacting RNAs (piRNAs). piRNAs show distinctive localization patterns in the genome, being predominantly grouped into 20-90-kilobase clusters, wherein long stretches of small RNAs are derived from only one strand. Similar piRNAs are also found in human and rat, with major clusters occurring in syntenic locations. Although their function must still be resolved, the abundance of piRNAs in germline cells and the male sterility of Miwi mutants suggest a role in gametogenesis.

To address the biological function of RNA interference (RNAi)-related pathways in mammals, we disrupted the gene Dicer1 in mice. Loss of Dicer1 lead to lethality early in development, with Dicer1-null embryos depleted of stem cells. Coupled with our inability to generate viable Dicer1-null embryonic stem (ES) cells, this suggests a role for Dicer, and, by implication, the RNAi machinery, in maintaining the stem cell population during early mouse development.