A bacterial Argonaute with noncanonical guide RNA specificity

Abstract

Eukaryotic Argonaute proteins induce gene silencing by small RNAguided recognition and cleavage of mRNA targets. Although structural similarities between human and prokaryotic Argonautes are consistent with shared mechanistic properties, sequence and structure-based alignments suggested that Argonautes encoded within CRISPR-cas [clustered regularly interspaced short palindromic repeats (CRISPR)-associated] bacterial immunity operons have divergent activities. We show here that the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) protein cleaves single-stranded target sequences using 5′-hydroxylated guide RNAs rather than the 5′-phosphorylated guides used by all known Argonautes. The 2.0-Å resolution crystal structure of an MpAgo-RNA complex reveals a guide strand binding site comprising residues that block 5′ phosphate interactions. Using structure-based sequence alignment, we were able to identify other putative MpAgo-like proteins, all of which are encoded within CRISPRcas loci. Taken together, our data suggest the evolution of an Argonaute subclass with noncanonical specificity for a 5′-hydroxylated guide.Argonaute | small noncoding RNA | RNA interference A rgonaute (Ago) proteins bind small RNA or DNA guides, which provide base-pairing specificity for recognition and cleavage of complementary nucleic acid targets. Members of this protein family are present in all three domains of life (1). In eukaryotes, Argonautes are the key effectors of RNA interference (RNAi) pathways that regulate posttranscriptional gene expression (2-4). However, the role of Argonaute proteins in bacteria and archaea, which lack RNAi pathways, remains poorly understood (5).Recent studies suggested that DNA-guided bacterial and archaeal Argonaute proteins are directly involved in host defense by cleaving foreign DNA elements, such as DNA viruses and plasmids (6, 7). In addition, a catalytically inactive Argonaute protein in Rhodobacter sphaeroides (RsAgo) was demonstrated to use RNA guides and possibly recruits an associated nuclease for subsequent target cleavage (8). Despite these divergent modes of action, bacterial and archaeal Argonaute proteins adopt a highly conserved bilobed architecture. Herein, an N-terminal and a PIWI-ArgonauteZwille (PAZ) domain constitute one lobe, whereas the other lobe consists of the middle (MID) domain and the catalytic RNase H-like P element-induced wimpy testis (PIWI) domain (9-15). Molecular structures of a eukaryotic Argonaute MID domain and an Archaeoglobus fulgidus Piwi (AfPiwi) enzyme bound to a guide RNA showed the importance of the 5′-terminal base identity, as well as the 5′ phosphate in guide strand binding, to Ago (10,[13][14][15][16][17]. Notably, recognition of the 5′ end of the guide in the MID domain and guide strand preorganization for target interaction are conserved across the entire Argonaute superfamily (1).The nucleic acid-guided binding and cleavage activities of Argonaute proteins are reminiscent of the activities of RNA-guided proteins within CRISPR-Cas systems [cluste...

DNA recognition by an RNA-guided bacterial Argonaute

“…Previous biochemical experiments showed that MpAgo binds RNA guides and preferentially targets DNA [12]. Analysis of the MpAgo ternary complex crystallized in this study revealed that the bound RNA-DNA heteroduplex adopts a B-form-like helical geometry.…”

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

“…While RsAgo is inactive as an endonuclease and our attempts to restore its catalytic activity by point mutagenesis were unsuccessful (Figure S3), pAgos from other species have guide-dependent endonuclease activity both in vitro and in vivo (Kaya et al, 2016; Swarts et al, 2014a, 2015; Wang et al, 2008). In contrast to CRISPR/Cas9, pAgo-gRNA complex can potentially target virtually any sequence of interest as efficient recognition of the target does not require the presence of Protospacer adjacent motif (PAM), a short sequence following the DNA sequence targeted by Cas9.…”

“…While eukaryotic Agos use gRNAs to recognize and cleave target RNAs (Liu et al, 2004), pAgos were shown to use gRNAs (e.g., Rhodobacter sphaeroides RsAgo and MpAgo) or DNAs (e.g., TtAgo) to recognize target DNAs (tDNAs) (Kaya et al, 2016; Makarova et al, 2009; Olovnikov et al, 2013; Swarts et al, 2014a, 2014b; Yuan et al, 2005). Functional studies suggested that pAgos defend genomes against invasive DNA (Hur et al, 2014; Olovnikov et al, 2013; Schirle et al, 2014; Swarts et al, 2014a, 2015), a function similar to their eukaryotic counterparts.…”

Structural Foundations of RNA Silencing by Argonaute

“…In accordance with this idea, the crystal structure of T. thermophilus Argonaute revealed that the guide seed region is displayed in a pre-organized helical conformation [68]. Subsequent structures have found that pre-ordering of at least the 5′ end (g2–g5) of the seed is feature of all prokaryotic and eukaryotic Argonaute proteins studied thus far [64, 65, 67, 83, 141–143]. Moreover, single molecule studies have demonstrated that Argonaute binds substantially faster and remains more stably associated to seed-paired target RNAs than an equivalent naked RNA duplex [6, 144].…”