Crystal Structure of Silkworm PIWI-Clade Argonaute Siwi Bound to piRNA

Abstract

PIWI-clade Argonaute proteins associate with PIWI-interacting RNAs (piRNAs) and silence transposable elements in animal gonads. Here, we report the crystal structure of a silkworm PIWI-clade Argonaute, Siwi, bound to the endogenous piRNA, at 2.4 Å resolution. Siwi adopts a bilobed architecture consisting of N-PAZ and MID-PIWI lobes, in which the 5' and 3' ends of the bound piRNA are anchored by the MID-PIWI and PAZ domains, respectively. A structural comparison of Siwi with AGO-clade Argonautes reveals notable differences in their nucleic-acid-binding channels, likely reflecting the distinct lengths of their guide RNAs and their mechanistic differences in guide RNA loading and cleavage product release. In addition, the structure reveals that Siwi and prokaryotic, but not eukaryotic, AGO-clade Argonautes share unexpected similarities, such as metal-dependent 5'-phosphate recognition and a potential structural transition during the catalytic-tetrad formation. Overall, this study provides a critical starting point toward a mechanistic understanding of piRNA-mediated transposon silencing.

Accommodation of Helical Imperfections in Rhodobacter sphaeroides Argonaute Ternary Complexes with Guide RNA and Target DNA

“…A similar conformational change is likely required for activation of catalysis by the eukaryotic PIWI-clade SIWI protein (Matsumoto et al, 2016). Plugged-in states are also observed in all available eukaryotic Ago2 complexes (Schirle et al, 2014).…”

“…Furthermore, phosphates 1 and 3 of the U1-U2 step of the bound guide are coordinated to a bound Mg 2+ , which in turn is bonded to the carboxylate of C-terminal Ago residue Leu777 (Figures 3A and 3B). This makes RsAgo similar to the PIWI-clade SIWI protein (Matsumoto et al, 2016) but distinguishes it from Ago-clade Agos (exemplified by hAgo2), in which corresponding contacts are formed by a conserved lysine residue (Elkayam et al, 2012; Schirle and MacRae, 2012). …”

“…In eukaryotes, Ago proteins form complexes with short interfering RNAs (siRNAs), microRNAs (miRNAs), and PIWI-interacting RNAs (piRNAs) that recognize complementary RNA targets resulting in mRNA degradation or translational and transcriptional inhibition (Bartel, 2004; Kuhn and Joshua-Tor, 2013; Parker, 2010). Structural biology studies on Ago proteins and their complexes with guide and target nucleic acids have greatly enhanced our understanding of gene silencing phenomena starting with studies of prokaryotic Agos (pAgos) and their complexes (Sheng et al, 2014; Song et al, 2004; Wang et al, 2009; Wang et al, 2008; Yuan et al, 2005) and subsequently extended to eukaryotic Agos and their complexes (Elkayam et al, 2012; Faehnle et al, 2013; Nakanishi et al, 2012, 2013; Schirle and MacRae, 2012; Schirle et al, 2014) and to PIWI proteins (Matsumoto et al, 2016). …”

“…Recent analysis of a PIWI-clade SIWI protein from silkworm revealed unexpected similarities with pAgos, including metal-dependent contacts with the RNA guide 5′ end in the MID pocket and similar organization of the active sites (Matsumoto et al, 2016). Further structural and functional studies of pAgos may shed light on the interference pathways mediated by these proteins and their eukaryotic counterparts and make a path to adopt pAgos as a tool for genome and epigenome engineering.…”

Hili Inhibits HIV Replication in Activated T Cells

“…Selected tRNAs are double stranded at their 5= and 3= ends. The distances between them are not sufficient to fit into the two separated RNA-binding pockets of the recently crystalized Piwil protein from silkworm (27). Nevertheless, Hili could potentially unwind the secondary structure of selected tRNAs to separate their 5= and 3= ends.…”