Argonaute (Ago) proteins are found in all three domains of life (bacteria, archaea, and eukaryotes). The best-characterized group is eukaryotic Ago (eAgo) proteins. Being the functional core of RNA interference machinery, eAgos are involved in the regulation of gene expression, silencing of mobile genome elements, and defence against viruses [1, 2]. From the structural and mechanistic point of view, all characterised eAgos are very similar, as they all use small (~ 21–30 nt) RNA molecules as guides for sequence-specific recognition of RNA targets, and are monomeric proteins sharing four conserved functional domains, which are organized in a bilobed structure [3]. The N-terminal lobe consists of N-domain that separates guide and target strands [4], and PAZ domain responsible for binding the 3′-terminus of the guide RNA; the C-terminal lobe consists of MID domain, which binds the 5′-terminus of the guide RNA, and PIWI domain, a nuclease [1, 2, 5, 6]. Upon recognition of the RNA target, eAgos may either cleave it employing catalytic activity of the PIWI domain or, particularly eAgo proteins that encode catalytically inactive PIWI domains, recruit partner proteins leading to degradation of the target RNA or repression of its translation [1, 7]. Target specificity of eAgos is determined solely by correct base pairing between guide and target RNA strands. Nevertheless, all eAgos associate only with 5’-phosphorylated guide RNAs, and many have intrinsic specificity for the 5′-terminal nucleotide of the guide RNA (gRNA). For example, A. thaliana AGO1, K. polysporus KpAgo, and H. sapiens hAgo2 prefer guide RNAs with a 5′-terminal uridine, A. thaliana AGO2 prefers 5′-A, and A. thaliana AGO5 prefers 5′-C [8–11]. The 5’-phosphate and the 5’-terminal nucleotide are recognized in conserved pockets of the MID domain.
Ago proteins are also identified in 9% and 32% of sequenced bacterial and archaeal genomes, respectively [12]. Unlike eAgos, prokaryotic Agos (pAgos) are diverse in terms of their structure, mechanism and function [6, 13–16]. The best understood are the so-called full-length or long pAgos, which are composed of N, PAZ, MID and PIWI domains, and thus closely resemble eAgos. There is mounting evidence that long pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids [1, 11]. However, unlike eAgos, which canonically use RNA guides for recognition of RNA targets in a process called RNA interference (RNAi) [17–19], different long pAgos may use either RNA or DNA guides and/or targets [11, 12, 16], and may associate with either phosphorylated (e.g. AaAgo, PfAgo, and RsAgo from Aquifex aeolicus, Pyrococcus furiosus, and Rhodobacter sphaeroides, respectively) [11] or non-phosphorylated (e.g., MpAgo, CbAgo, LrAgo, KjAgo from Marinitoga piezophila, Clostridium butyricum, Limnothrix rosea, Kordia jejudonensis, respectively) [11, 20, 21] guide strands. Interestingly, recently described KmAgo from Kurthia massiliensis can utilize both DNA and RNA guides to cleave DNA and RNA targets in vitro, albeit with different efficiencies [22, 23]. Some long pAgos also specifically recognize the 5’-terminal nucleotide of the guide strand, e.g., CbAgo from Clostridium butyricum prefers 5’-terminal deoxyadenosine, and TtAgo prefers 5’-terminal dC [11, 24], while RsAgo from Rhodobacter sphaeroides prefers guide RNA with 5’-U [25, 26]. The majority (~ 60%) of identified pAgos are ‘short’, as they encode just MID and PIWI domains, the latter being catalytically inactive due to active site mutations. The mechanism, guide/target preferences and function of short pAgos is an emerging topic in the Argonaute field, as evidenced by recent characterization of SPARTA and GsSir2/Ago antiviral systems [13, 14, 27].
In this work we focus on the short (alternatively classified as a truncated long-B [6]) prokaryotic Argonaute AfAgo (also known as AfPIWI or Af1318 [28, 29]) encoded by a hyperthermophilic archaeon Archaeoglobus fulgidus [6]. Like a typical short pAgo, AfAgo contains only MID and catalytically inactive PIWI domains. Even though AfAgo is one of the first and one of the best structurally characterized prokaryotic Argonautes, with an apo-, DNA-, and two RNA-bound structures currently available [28–31], its guide/target preferences remain undefined. We show here that AfAgo co-purifies with small RNA molecules carrying 5′-terminal AU nucleotides in vivo and investigate its affinity to various RNA and DNA guide/target strands in vitro. We also present X-ray structures of AfAgo bound to DNA oligoduplexes with 5’-ATT and 5’-ATC terminal sequences that provide structural details on the base-specific AfAgo interactions with the termini of both guide and target strands. Our findings broaden the range of currently known Argonaute-nucleic acid recognition mechanisms.