Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein

Abstract

Argonaute proteins (AGOs) are essential effectors in RNAmediated gene silencing pathways. They are characterized by a bilobal architecture, in which one lobe contains the N-terminal and PAZ domains and the other contains the MID and PIWI domains. Here, we present the first crystal structure of the MID-PIWI lobe from a eukaryotic AGO, the Neurospora crassa QDE-2 protein.Compared to prokaryotic AGOs, the domain orientation is conserved, indicating a conserved mode of nucleic acid binding. The PIWI domain shows an adaptable surface loop next to a eukaryote-specific α-helical insertion, which are both likely to contact the PAZ domain in a conformation-dependent manner to sense the functional state of the protein. The MID-PIWI interface is hydrophilic and buries residues that were previously thought to participate directly in the allosteric regulation of guide RNA binding. The interface includes the binding pocket for the guide RNA 5′ end, and residues from both domains contribute to binding. Accordingly, micro-RNA (miRNA) binding is particularly sensitive to alteration in the MID-PIWI interface in Drosophila melanogaster AGO1 in vivo. The structure of the QDE-2 MID-PIWI lobe provides molecular and mechanistic insight into eukaryotic AGOs and has significant implications for understanding the role of these proteins in silencing.P roteins of the Argonaute (AGO) family play essential roles in RNA-mediated gene silencing mechanisms in eukaryotes (1, 2). They are loaded with small noncoding RNAs to form the core of RNA-induced silencing complexes, which repress the expression of target genes at the transcriptional or posttranscriptional level (1, 2). The targets to be silenced are selected through base-pairing interactions between the loaded small RNA (also known as the guide RNA) and an mRNA target containing partially or fully complementary sequences (1-3).Thus far, structural information on full-length AGOs has been available only for the homologous proteins from Archaea and Eubacteria, which preferentially use DNA as a guide (4-10). These studies revealed that AGOs consist of four domains: the N-terminal domain; the PAZ domain, which binds the 3′ end of guide RNAs/DNAs; the MID domain, which provides a binding pocket for the 5′ phosphate of guide RNAs/DNAs; and the PIWI domain, which adopts an RNase H fold and has endonucleolytic activity in some, but not all, AGOs (4-11).For the eukaryotic AGO clade of Argonaute proteins, structural information is available only for the isolated PAZ domains of Drosophila melanogaster (Dm) AGO1 and AGO2, human AGO1 (12-16) and the MID domains of human AGO2 and Neurospora crassa (Nc) QDE-2 (17, 18). Structural information is also available for PAZ domains of the PIWI clade of AGOs (19,20). These studies showed that the PAZ and MID domains of eukaryotic AGOs adopt folds similar to the prokaryotic homologs and recognize the 3′-and 5′-terminal nucleotides of the guide strand, respectively, in a similar manner to their prokaryotic counterparts (12-18).Our previous structure of the isolate...

The MID-PIWI module of Piwi proteins specifies nucleotide- and strand-biases of piRNAs

“…A likely explanation for this is the absence of the adjacent PIWI domain, which is known to contribute to the nucleotide-binding pocket in Neurospora crassa QDE-2 MID-PIWI and full-length eukaryotic Argonaute structures (Boland et al 2011;Nakanishi et al 2012;Schirle and MacRae 2012). In fact, Neurospora crassa QDE-2 MID-PIWI domain is required to bind RNA, while the MID domain alone is insufficient (Boland et al 2011). Therefore, we proceeded by positioning the ligands (NMPs) in the MIWI MID structure by docking analysis.…”

“…Substitution of the tyrosine in Bombyx Siwi with glutamate (Y607E) or extension of the C-terminal end by a single alanine (A) residue (C-term +A), which presumably affects the C terminus's contribution to 5 ′ phosphate recognition within the MID domain (Boland et al 2011;Nakanishi et al 2012;Schirle and MacRae 2012), were found to drastically reduce small RNA binding in vitro (Kawaoka et al 2011). Mutation within the MID domain in AGO clade members is also shown to drastically reduce small RNA binding in vivo (Boland et al 2011;Rudel et al 2011). Taken together, this confirms that 5 ′ phosphate recognition within the Piwi MID domain is essential for piRNA biogenesis in vivo and that the MID environment in some proteins may further specify the 5 ′ -end nucleotide bias of the bound piRNA.…”

Dicer is dispensable for asymmetric RISC loading in mammals

“…This structural conformation should favor the orientation of the small RNA duplex with the end that is more easily unwound (i.e., the less stable end) toward the phosphate-binding pocket. The high homology of the MID and PIWI domains, in particular the architecture of the C-terminal carboxylate that forms the phosphate-binding pocket, suggests that the binding mode for a small RNA molecule is commonly shared by Ago proteins (Parker et al 2005;Wang et al 2008;Boland et al 2011). In the case of fly Ago2-RISC assembly, Dcr-2/R2D2 binding might double-check or amplify the sensing of thermodynamic asymmetry in addition to Ago2 itself; an siRNA duplex needs to bind Dcr-2/R2D2 before entering Ago2, and yet the duplex might transiently shuttle between Dcr-2/R2D2 and Ago2, with each protein checking the asymmetry, before it is properly docked into Ago2.…”

RNA silencing in Monterey

“…However, Filipp Frank (Nahum Sonenberg and Bhushan Nagar laboratories, McGill University, Canada) reported that nucleotides and cap analogs readily interact with the hAGO2 MID domain through the miRNAbinding pocket, but that additional nucleotide interactions outside this region were not observed (Frank et al, 2011). Elisa Izaurralde (Max Planck Institute for Developmental Biology, Germany) presented new work on the MID-PIWI lobe of QDE-2 (Boland et al, 2011). Siwi also stably bound longer substrates with 1U preference.…”