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

• Published in: Nature 2005-03, Vol.434 (7033), p.666-670
• Main Authors: Patel, Dinshaw J, Ma, Jin-Biao, Yuan, Yu-Ren, Meister, Gunter, Pei, Yi, Tuschl, Thomas
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Archaea; Archaeal Proteins - chemistry; Archaeal Proteins - metabolism; Archaeoglobus fulgidus; Archaeoglobus fulgidus - chemistry; Argonaute Proteins; Base Sequence; Binding Sites; Biological and medical sciences; Cellular biology; Crystallography; Eukaryotic Initiation Factor-2; Fundamental and applied biological sciences. Psychology; Humanities and Social Sciences; Humans; letter; Marine; Models, Molecular; Molecular biophysics; Molecular Sequence Data; multidisciplinary; Nucleic Acid Conformation; Peptide Initiation Factors - chemistry; Peptide Initiation Factors - genetics; Peptide Initiation Factors - metabolism; Phosphates - metabolism; Protein Structure, Tertiary; Proteins; Ribonucleic acid; RNA; RNA Interference; RNA, Double-Stranded - chemistry; RNA, Double-Stranded - genetics; RNA, Double-Stranded - metabolism; RNA, Guide - chemistry; RNA, Guide - genetics; RNA, Guide - metabolism; RNA, Messenger - chemistry; RNA, Messenger - genetics; RNA, Messenger - metabolism; Science; Science (multidisciplinary); Structure in molecular biology; Substrate Specificity; Tridimensional structure
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature03514

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.