Structural basis for 5'-end-specific recognition of guide RNA by the A. fulgidus Piwi protein.
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.
Ma
Jin-Biao
JB
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Yuan
Yu-Ren
YR
Meister
Gunter
G
Pei
Yi
Y
Tuschl
Thomas
T
Patel
Dinshaw J
DJ
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D019611:Archaeoglobus fulgidus / Q000737:chemistry*; D060565:Argonaute Proteins; D001483:Base Sequence; D001665:Binding Sites; D015852:Eukaryotic Initiation Factor-2; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D058727:RNA, Small Untranslated
C466422:AGO2 protein, human; D019843:Archaeal Proteins; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D010448:Peptide Initiation Factors; D010710:Phosphates; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D058727:RNA, Small Untranslated