Argonaute proteins and small interfering RNAs (siRNAs) are the known signature components of the RNA interference effector complex RNA-induced silencing complex (RISC). However, the identity of "Slicer," the enzyme that cleaves the messenger RNA (mRNA) as directed by the siRNA, has not been resolved. Here, we report the crystal structure of the Argonaute protein from Pyrococcus furiosus at 2.25 angstrom resolution. The structure reveals a crescent-shaped base made up of the amino-terminal, middle, and PIWI domains. The Piwi Argonaute Zwille (PAZ) domain is held above the base by a "stalk"-like region. The PIWI domain (named for the protein piwi) is similar to ribonuclease H, with a conserved active site aspartate-aspartate-glutamate motif, strongly implicating Argonaute as "Slicer." The architecture of the molecule and the placement of the PAZ and PIWI domains define a groove for substrate binding and suggest a mechanism for siRNA-guided mRNA cleavage.

Genetic, biochemical and structural studies have implicated Argonaute proteins as the catalytic core of the RNAi effector complex, RISC. Here we show that recombinant, human Argonaute2 can combine with a small interfering RNA (siRNA) to form minimal RISC that accurately cleaves substrate RNAs. Recombinant RISC shows many of the properties of RISC purified from human or Drosophila melanogaster cells but also has surprising features. It shows no stimulation by ATP, suggesting that factors promoting product release are missing from the recombinant enzyme. The active site is made up of a unique Asp-Asp-His (DDH) motif. In the RISC reconstitution system, the siRNA 5' phosphate is important for the stability and the fidelity of the complex but is not essential for the creation of an active enzyme. These studies demonstrate that Argonaute proteins catalyze mRNA cleavage within RISC and provide a source of recombinant enzyme for detailed biochemical studies of the RNAi effector complex.

RISC, the RNA-induced silencing complex, uses short interfering RNAs (siRNAs) or micro RNAs (miRNAs) to select its targets in a sequence-dependent manner. Key RISC components are Argonaute proteins, which contain two characteristic domains, PAZ and PIWI. PAZ is highly conserved and is found only in Argonaute proteins and Dicer. We have solved the crystal structure of the PAZ domain of Drosophila Argonaute2. The PAZ domain contains a variant of the OB fold, a module that often binds single-stranded nucleic acids. PAZ domains show low-affinity nucleic acid binding, probably interacting with the 3' ends of single-stranded regions of RNA. PAZ can bind the characteristic two-base 3' overhangs of siRNAs, indicating that although PAZ may not be a primary nucleic acid binding site in Dicer or RISC, it may contribute to the specific and productive incorporation of siRNAs and miRNAs into the RNAi pathway.

Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.

The observed nucleotide-dependent conformational changes in HslU and their governing principles provide a framework for the mechanistic understanding of other AAA(+) proteins.

Argonaute is a key enzyme of various RNA silencing pathways. We use single-molecule fluorescence measurements to characterize the reaction mechanisms of the core-RISC (RNA-induced silencing complex) composed of human Argonaute 2 and a small RNA. We found that target binding of core-RISC starts at the seed region, resulting in four distinct reaction pathways: target cleavage, transient binding, stable binding, and Argonaute unloading. The target cleavage requires extensive sequence complementarity and dramatically accelerates core-RISC recycling. The stable binding of core-RISC is efficiently established with the seed match only, providing a potential explanation for the seed-match rule of miRNA (microRNA) target selection. Target cleavage on perfect-match targets sensitively depends on RNA sequences, providing an insight into designing more efficient siRNAs (small interfering RNAs).

WDR5 is a component of the mixed lineage leukemia (MLL) complex, which methylates lysine 4 of histone H3, and was identified as a methylated Lys-4 histone H3-binding protein. Here, we present a crystal structure of WDR5 bound to an MLL peptide. Surprisingly, we find that WDR5 utilizes the same pocket shown to bind histone H3 for this MLL interaction. Furthermore, the WDR5-MLL interaction is disrupted preferentially by mono-and di-methylated Lys-4 histone H3 over unmodified and tri-methylated Lys-4 histone H3. These data implicate a delicate interplay between the effector, WDR5, the catalytic subunit, MLL, and the substrate, histone H3, of the MLL complex. We suggest that the activity of the MLL complex might be regulated through this interplay.DNA in eukaryotic cells forms into higher order structures made up of chromatin. Chromatin, and its higher order structure, is dynamically modified in concert with gene expression and cell cycle. The fundamental unit of the chromatin, the nucleosome, is composed of a histone octamer (dimers of H2A, H2B, H3, and H4) and 146 base pairs of DNA, which wrap around the histone octamer (1). Histones have unstructured N-or C-terminal tails protruding out of the nucleosome core structure. These tails are covalently modified with methyl, acetyl, phospho, ubiquitin, sumo, and ADP-ribose moieties (2).Addition and removal of these covalent modifications can affect gene expression, as the modifications can serve to recruit effector proteins. Methylation of lysine (or arginine) of histones adds another layer of complexity, as lysine can be mono-, di-, and tri-methylated, and arginine mono-, and di-methylated. Histone H3 Lys-4 and Lys-27 methylations are particularly interesting, because these marks are antagonistic to each other in their functions in that they are highly correlated with gene activation (Lys-4) and repression (Lys-27) (3).Previously, in an effort to find an effector molecule recognizing a methylated Lys-4 of histone H3, the WDR5 protein was identified as a methyl Lys-4 H3-specific-binding protein, and it was shown that WDR5 is required for tri-methylation of histone H3 in vivo (4). WDR5 belongs to the WD40 repeat protein family. The WD40 repeat is a well characterized protein-protein interaction domain involved in diverse cellular processes. The WDR5 protein was also previously identified as a component of mixed lineage leukemia (MLL) 3 complex (5). The MLL complex is a histone H3 Lys-4 methyltransferase. ASH2L, RBBP5, and MLL interact with WDR5 to comprise a core MLL complex (5). WDR5, ASH2L, and RBBP5 also form complexes with the Set1 protein and with the four isoforms of MLL: MLL1, MLL2, MLL3, and MLL4. The MLL and Set1 proteins are the catalytic subunits of these complexes, and each contains a SET domain at the C terminus. However, the molecular functions of each component are largely unknown, as are many of the molecular mechanisms that regulate the function of the MLL complex.WDR5 is a component of the MLL complex, is required for histone H3 tri-methylation, and bin...

p55 is a common component of many chromatin-modifying complexes and has been shown to bind to histones. Here, we present a crystal structure of Drosophila p55 bound to a histone H4 peptide. p55, a predicted WD40 repeat protein, recognizes the first helix of histone H4 via a binding pocket located on the side of a ␤-propeller structure. The pocket cannot accommodate the histone fold of H4, which must be altered to allow p55 binding. Reconstitution experiments show that the binding pocket is important to the function of p55-containing complexes. These data demonstrate that WD40 repeat proteins use various surfaces to direct the modification of histones.Supplemental material is available at http://www.genesdev.org.Received January 18, 2008; revised version accepted March 13, 2008. In eukaryotic cells, DNA is hierarchically packaged into higher order structures called chromatin. The basic unit of chromatin is the nucleosome, which is formed from 146 base pairs of DNA wrapped around a histone octamer. Chromatin is a dynamic structure and can be modified in several ways (Li et al. 2007). First, chromatin is actively assembled and disassembled by histone chaperone complexes. These assembly and disassembly processes are tightly coupled with DNA replication and gene expression (Groth et al. 2007). Second, chromatin can be covalently modified; N-or C-terminal tails of histones can be methylated, acetylated, phosphorylated, adenylated, or ubiquitylated. These modifications can be utilized as marks for recruiting effector proteins and might also directly alter chromatin folding. Third, chromatin structure is altered by ATP-dependent chromatin remodeling complexes, which can alter DNA accessibility by disrupting DNA-histone contacts.p55 (p55 or Nurf55 in fly, RbAp48/46 in human, and MSI1 in plants) is highly conserved from plants to human. RbAp48/46 was initially identified as a retinoblastoma-associated protein (Huang et al. 1991;Qian et al. 1993). Subsequent studies showed that p55 is a common component of many different chromatin-modifying complexes with a variety of functions. p55 is the smallest subunit in the Chromatin Assembly Factor 1 (CAF1) complex as well as a component of the ATP-dependent chromatin remodeling complexes-Nucleosome Remodeling Factor (NURF), and Nucleosome Remodeling and Deacetylase (NuRD) (Smith and Stillman 1989;Tyler et al. 1996;Verreault et al. 1996;Martinez-Balbas et al. 1998;Wade et al. 1998;Zhang et al. 1998). p55 is a component of acetyltransferase (Hat1), histone deacetylase (HDAC1), and histone methyltransferase (Polycomb-Repressive Complex2, PRC2) complexes, and it has been shown that p55 is critical for the function of these complexes (Parthun et al. 1996;Taunton et al. 1996;Hassig et al. 1997;Zhang et al. 1997;Verreault et al. 1998;Czermin et al. 2002;Kuzmichev et al. 2002;Muller et al. 2002). Furthermore p55 copurifies with additional complexes involved in gene regulation (Zhang et al. 1997;Korenjak et al. 2004).Depleting p55 causes a variety of epigenetic defects (Lu and Horvitz 1...