Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

Abstract

RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism 1-3 mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold 4,5 critical for the endonuclease cleavage activity of RISC 4-6 . Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5′-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5′ end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5′ end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5′ phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-RNA complex, and that determined elsewhere 7 , provide direct support for the 5′ region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5′ end of the guide RNA.Small interfering RNAs (siRNAs), the cleavage products of RNase III enzyme Dicer, consist of RNA duplexes that contain two-nucleotide 3′ overhangs and 5′-phosphorylated ends. These unique features at the termini of 19-23-base-pair duplexes, which distinguish siRNAs from other endogenous RNAs, are also required for their functional role in RNAi-mediated processes. Extensive structural and biochemical studies on PAZ (for PIWI/Argonaute/ Zwille)-RNA recognition [8][9][10] , culminating in the structures of PAZ bound to singlestranded RNA 11 and siRNA-like duplexes 12 , have established that the 3′ overhang isCorrespondence and requests for materials should be addressed to D.J.P. (pateld@mskcc.org).. Supplementary Information accompanies the paper on www.nature.com/nature. Competing interests statementThe authors declare that they have no competing financial interests.Coordinates have been deposited in the Protein Data Bank under accession code 1YTU. Here we describe the 2.5-Å crystal structure of the A. fulgidus Piwi protein (Fig. 1a) bound to a 5′-phosphate-containing 21-mer RNA, capable of self-complementary duplex formation (Fig. 1b). The crystallographic asymmetric unit contains two Piwi proteins, each bound to a short four-base-pair segment of duplex, positioned adjacent to the 5′ phosphate and its attached unpaired nucleotide (Fig. 1c). The structure of the A. fulgidus Piwi protein, which consists of a short N-terminal element and two domains labelled A and B (Fig. 1a), is similar (root-mean-square deviation = 0.65Å for Cα) in the free state 5 and in the RNAbound complex. The RNA, whose sequence ...

Determinants of specific RNA interference-mediated silencing of human β-globin alleles differing by a single nucleotide polymorphism

“…This result suggests that the Ago2 catalytic site can accommodate some siRNA:mRNA mismatches, in particular, combinations of mismatches that involve the smaller pyrimidine residues. There are no structural studies of Ago2, but two groups have crystallized the endonuclease domain of a homologous bacterial protein bound to a small siRNA-like duplex (17,18). Although the 5Ј end of the siRNA guide strand was well ordered in the crystal and embedded in the bacterial Piwi protein, the region corresponding to position 10 was disordered, suggesting that the active site may allow some flexibility for binding.…”

Principles and effects of microRNA-mediated post-transcriptional gene regulation

“…The three-dimensional structures of bacterial and archaeal Argonaute proteins and the PAZ domains of eucaryotic Argonautes revealed that the PAZ domain binds the short single-stranded 3 0 'tails' characteristic of an siRNA. It has been postulated that the PAZ domain initiates incorporation of a small RNA into RISC by binding the single-stranded 2nt 3 0 overhang of an siRNA or miRNA duplexes (Lingel et al, 2003;Song et al, 2003Song et al, , 2004Yan et al, 2003;Ma et al, 2005). More remarkably, these structural studies revealed that the key to understanding the sequence-specific activity of RISC resides in the structure of the PIWI domain, whose three-dimensional fold clearly places in the RNase H enzyme family of nucleic-acid-directed RNA-endonucleases Ma et al, 2005;Parker et al, 2005;Yuan et al, 2005).…”

“…It has been postulated that the PAZ domain initiates incorporation of a small RNA into RISC by binding the single-stranded 2nt 3 0 overhang of an siRNA or miRNA duplexes (Lingel et al, 2003;Song et al, 2003Song et al, , 2004Yan et al, 2003;Ma et al, 2005). More remarkably, these structural studies revealed that the key to understanding the sequence-specific activity of RISC resides in the structure of the PIWI domain, whose three-dimensional fold clearly places in the RNase H enzyme family of nucleic-acid-directed RNA-endonucleases Ma et al, 2005;Parker et al, 2005;Yuan et al, 2005). This finding is consistent with earlier biochemical studies showing that chemistry of cleavage by human and fly RISC has many similarities to RNAse H-mediated catalysis: a requirement for the divalent cation Mg 2 þ and cleavage products with 5 0 terminal phosphates and 3 0 terminal hydroxyl groups (Martinez and Tuschl, 2004;Schwarz et al, 2004).…”

Control of translation and mRNA degradation by miRNAs and siRNAs: Table 1.

“…The Argonaute protein family is diverse, with all members containing a PAZ domain, which is involved in miRNA/siRNA binding, and a PIWI domain, which is related to RNaseH endonucleases and functions in slicer activity (Lingel and Sattler 2005). Argonaute proteins directly interact with the miRNA/siRNA (Song et al 2003;Ma et al 2004Ma et al , 2005. Most eukaryotes examined contain multiple Argonaute family members, with different Argonautes often specialized for distinct functions.…”