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 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 critical for the endonuclease cleavage activity of RISC. 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, 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.

Figure 1

Crystal structure of the A. fulgidus Piwi–RNA complex. a, The A. fulgidus Piwi protein consists of a yellow N-domain (1–37), a magenta domain A (38–167) and a cyan domain B (168–427). b, The sequence and pairing alignment of the 21-mer RNA. The red and green segments are observed in the crystal structure of the complex, whereas those in black are disordered. c, The relative alignments of the two Piwi proteins together with their bound RNAs within the asymmetric unit. d, e, Ribbon (d) and electrostatic (e) views of the complex. The RNA is shown in a stick representation.

Figure 2

The 5′-phosphate-binding site in the A. fulgidus Piwi–RNA complex. a, Positioning of the 5′ phosphate in a basic pocket lined by residues of domain A (K127, Y123, Q137, Q159 and K163), domain B (C-terminal L427) and a bound divalent cation in orange. Bases A1 and G2 are splayed apart, with unpaired A1 stacked on Y123. b, The octahedral coordination of the bound divalent cation, presumably Mg²⁺, involving protein and RNA oxygens and a water molecule. c, Positioning of the short four-base-pair duplex segment involving the G2-A3-C4-A5 segment (red) with its complementary partner (green). Note intermolecular hydrogen bonds to the non-bridging phosphate oxygens of the A1-G2-A3-C4-A5 segment.

Figure 3

Mutation studies of conserved 5′-phosphate-binding residues in A. fulgidus Piwi protein and human Ago2 protein. a, Sequence alignment of conserved 5′-phosphate-binding residues across various species of Piwi and Argonaute proteins. Prefix Hs, human; Dm, Drosophila; Aa, Aquifex aeolicus, Af, Archaeoglobus fulgidus. b, Representative binding curves (average of three runs) for A. fulgidus Piwi single-site mutants with 21-nucleotide RNA labelled with 5′-³²P by double-filter binding assay. The deduced K_d,app values are listed below. c, The corresponding binding curves for A. fulgidus PIWI dual-site mutants. d, The cleavage activities of wild-type human Ago2 and its mutants. The black bar at the right of the image represents the region of the target RNA complementary to the siRNA used. The expression levels of the proteins used in the assays were assessed by western blotting with anti-HA antibody and are shown in the lower panel.

Figure 4

Model of A. fulgidus Piwi protein (Af Piwi) bound to an 18-base-pair RNA duplex with 5′-phosphorylated single-nucleotide overhangs. a, The 5′-end nucleotide and four-base-pair duplex (coloured in red and green for guide strand and target strand, respectively) observed in the crystal structure of the complex were extended by 14-base-pair A-form duplex (coloured in tan and white for guide strand and target strand, respectively). The 5′ end anchoring pocket and putative catalytic site are circled, and the phosphate cleavage site is marked by a red ball. b, Electrostatic surface view of the model of the Af Piwi–dsRNA complex.