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RNA Biol
RNA Biol
RNA Biology
1547-6286
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10.1080/15476286.2024.2380948
2380948
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Review Article
Review
Structures and functions of short argonautes
C. WANG ET AL.
RNA BIOLOGY
https://orcid.org/0000-0002-0508-0084
Wang Chen a b *
https://orcid.org/0000-0002-3707-2265
Shen Zhangfei a b *
https://orcid.org/0000-0002-9763-1196
Yang Xiao-Yuan a b c
https://orcid.org/0000-0002-6281-1752
Fu Tian-Min a b c
a Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University , Columbus, OH, USA
b The Ohio State University Comprehensive Cancer Center , Columbus, OH, USA
c Program of OSBP, The Ohio State University , Columbus, OH, USA
CONTACT Chen Wang Wang.17764@osu.edu
Tian-Min Fu Fu.978@osu.edu Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH, USA
* These authors contributed equally to this work.

1 9 2024
2024
1 9 2024
21 1 17
Integra30 8 2024
Integra30 8 2024
03 7 2024
08 7 2024
© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Argonaute proteins (Agos) represent a highly conserved family of proteins prevalent in all domains of life and have been implicated in various biological processes. Based on the domain architecture, Agos can be divided into long Agos and short Agos. While long Agos have been extensively studied over the past two decades, short Agos, found exclusively in prokaryotes, have recently gained attention for their roles in prokaryotic immune defence against mobile genetic elements, such as plasmids and phages. Notable functional and structural studies provide invaluable insights into the underlying molecular mechanisms of representative short Ago systems. Despite the diverse domain arrangements, short Agos generally form heterodimeric complexes with their associated effector proteins, activating the effector’s enzymatic activities upon target detection. The activation of effector proteins in the short Ago systems leads to bacterial cell death, a mechanism of sacrificing individuals to protect the community.

Keywords

Argonaute
SPARTA
SPARSA
SPARDA
The author(s) reported that there is no funding associated with the work featured in this article.
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pmcIntroduction

First identified in eukaryotes, Argonaute proteins (Agos) represent an ancient and broadly conserved family of proteins prevalent in all domains of life [1–4]. Agos employ single-stranded oligonucleotides, typically around 15 to 30 nucleotides (nt) in length, as guides to engage with complementary nucleic acid targets, playing pivotal roles in various biological processes including post-transcriptional gene silencing and host defence [5–14].

Based on the domain architecture, Agos can be divided into two categories: long Agos and short Agos [2,15,16] (Figure 1A–C). Long Agos are composed of four major domains, namely the N-terminal (N), PIWI-Argonaute-Zwille (PAZ), middle (MID) and P-element-induced wimpy testis (PIWI) domains (N-PAZ-MID-PIWI), along with two domain linkers, linker 1 (L1) and linker 2 (L2) [1,2,17] (Figure 1A). N-PAZ and MID-PIWI domains form a bilobal structure, which accommodates the nucleic acids (guide-target duplex) in-between the lobes. In the N-PAZ lobe, the N domain acts as a wedge, assisting in the unwinding of the duplex; the PAZ domain facilitates the binding of the 3’-end of the guide through a hydrophobic pocket [1,2,17]. Within the MID-PIWI lobe, the MID domain recognizes the 5′-end of the guide, while the PIWI domain adopts a characteristic RNase H fold with a conserved DEDX (where X is D, H, or K) tetrad and functions to cleave target nucleic acids [1,2,17]. Notably, some long Agos contain a catalytically inactive PIWI (PIWI*, which lacks a catalytic tetrad) domain and lost the ability to cleave nucleic acid targets [15,16,18]. Eukaryotic Agos bind small RNA guides to recognize and silence RNA targets, whereas most known prokaryotic long Agos preferentially target DNA [3,16]. For example, Clostridium butyricum Ago (CbAgo) was shown to target both plasmid DNA and phage genomes, thereby serving as an anti-plasmid and anti-phage defence module [11]. Figure 1. Schematic domain architectures of long Ago and short Ago systems.

(A) Schematic domain architecture of a long Ago and a short Ago. NTD, N-terminal domain. L1, linker 1. PAZ, PIWI-Argonaute-Zwille. L2, linker 2. MID, middle. PIWI, P-element-induced wimpy testis. PIWI*, catalytically inactive PIWI that lacks a catalytic tetrad. (B) Schematic domain architecture of short Agos systems. Short Ago systems can be divided into different clades based on the features of their associated APAZ-containing effector proteins and effector domains. SPARSA, short prokaryotic Argonaute/SIR2-APAZ, also known as Sir2/Ago. SPARTA, short prokaryotic Argonaute/TIR-APAZ. SPARDA, short prokaryotic Argonaute and DNase/RNase-APAZ. SiAgo system, the short Ago system from Sulfolobus islandicus which comprises a short Ago and two effector proteins [SiAgo-associated protein 1 (SiAga1) and membrane protein SiAga2]. (C) Maximum likelihood-based unrooted phylogenetic tree containing selected eukaryotic Agos (eAgos) and all prokaryotic Ago (pAgo) homologs identified in the RefSeq database, including Sulfolobus islandicus pAgo (SiAgo) homologs. [Adapted from Koopal, Mutte and Swarts (2023)]. Short pAgos are classified into two major clades: Clade S1 and Clade S2. Each clade contains two sub-clades, as illustrated in the phylogenetic tree.

In contrast to long Agos, short Agos lack the N and PAZ domains and consist of the MID and PIWI* domains with no catalytic activity (Figure 1B) [1,15,16]. Short Agos are typically encoded in the same operon with (or fused to) accessory effector proteins featuring an “analog of PAZ” (APAZ) domain that is homologous to the N and L1 domains of long Agos [1,15,16]. Over the past two decades, the structures and functions of long Agos have been extensively characterized, while short Agos remain largely uncharted territory until recently. Recent studies revealed that the short Ago systems are implicated in defence against mobile genetic elements via an abortive infection (Abi) response [19–24]. In this review, we highlight recent progress in the structural and functional characterization of several short Agos systems.

Overview of short argonautes

Despite phylogenetic diversity, short Agos can be generally divided into four clades – S1A, S1B, S2A and S2B – based on the features of their effector proteins [16,19,23] (Figure 1B–C). Across all clades, short Agos clustered or fused with an APAZ domain-containing effector protein that is encoded within the same operon. The effector protein usually contains an N-terminal (putative) enzymatic domain followed by a C-terminal APAZ domain (Figure 1B) [16,19,23]. The short Ago together with its effector protein constitutes a “short Ago system”. These short Ago systems are named based on the N-terminal domains of the effector proteins [16]. Specifically, in S1A and S1B clades, the effector proteins contain a Silent Information Regulator 2 (SIR2, also known as Sirtuin) domain. Thus, the short Ago systems in these two clades were named short prokaryotic Argonaute/SIR2-APAZ (SPARSA, also known as Sir2/Ago). As there is a Toll-Interleukin Receptor (TIR) domain in clade S2A, these systems were therefore referred to as short prokaryotic Argonaute/TIR-APAZ (SPARTA). Within S2B subclades, some systems consist of DNase or RNase domains and were coined as short prokaryotic Argonaute and DNase/RNase-APAZ (SPARDA) [23]. Apart from these systems, a peculiar short Ago system that does not belong to the four clades mentioned above is the Sulfolobus islandicus Ago (SiAgo) system, which comprises a short Ago and two accessory proteins, SiAgo-associated protein 1 (SiAga1, which structurally corresponds to the N-L1-L2 part of long pAgos) and membrane protein SiAga2 (Figure 1B) [16,22,25,26].

Biogenesis of guide oligonucleotides

Eukaryotic Agos use microRNAs (miRNA) as guides, which are transcribed by RNA polymerase and processed by Drosha and Dicer [27]. In contrast, guides used by prokaryotic Agos derive from variable sources. For example, both TtAgo and CbAgo, long prokaryotic Agos, use DNA as guides [11,28]. TtAgo can generate its own guides by chopping invading DNA, while CbAgo relies on RecBCD to generate its guides of plasmid origin [11,28]. RsAgo, another long prokaryotic Ago, uses small interfering RNA guides that derived from degraded mRNA [29]. Similar to RsAgo, all the characterized short Agos use RNA as guides. However, the biogenesis of guides for short Agos remains unclear. In vivo pulldown assay combined with sequencing of SPARTA revealed that guide RNAs of SPARTA correlate with the abundance of mRNAs, suggesting that the guides for SPARTA may derive from RNA transcription in bacteria [19]. How the guide RNAs are processed remains to be explored.

Functions of short argonautes

Recent breakthrough studies have yielded valuable insights into the roles of short Ago systems in prokaryotic immune defence against mobile genetic elements [19,20,22–24]. SPARTA, SPARSA, SPARDA and SiAgo systems stand as exemplary instances within the defence-related short Ago systems, in which the short Agos maintain the ability for guide-mediated nucleic acid binding and function with a variety of accessory effector proteins that induce bacterial cell death upon target detection [19,20,22–24].

Both SPARTA and SPARSA systems consist of a short Ago and an effector protein containing a NADase domain (TIR-APAZ and SIR2-APAZ, respectively). These components form complexes that are capable of depleting nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) upon binding to invader DNA [19,20] (Figure 2A). SPARTA systems from Crenotalea thermophila (CrtSPARTA) and Maribacter polysiphoniae (MapSPARTA) degrade NAD+ (and NADP+) to trigger bacterial cell death upon plasmid invasion [19]. Specifically, the TIR-APAZ protein and short Ago form a heterodimer for RNA-guided target DNA binding. Upon RNA-DNA duplex binding, SPARTA assembles into a tetramer with the TIR domain converting NAD(P)+ to noncyclic adenosine diphosphate ribose (phosphate) [ADPR(P)] and nicotinamide (NAM). The depletion of NAD(P)+ by SPARTA eventually leads to bacterial cell death [19]. Figure 2. Schematic of working mechanisms of short Ago systems.

(A) SPARTA, SPARSA and SPARDA systems encode a short Ago protein together with an APAZ-containing effector protein with a (putative) catalytic domain, respectively. In these systems, the short Ago forms a heterodimer with its associated effector protein. Directed by RNA guides, this Ago-effector complex senses invading DNA, thus activating the effector protein’s enzymatic activities and eventually resulting in bacterial cell death. (B) The SiAgo system contains a short Ago protein and two effector proteins (Aga1 and Aga2). In this system, the short Ago and Aga1 form a heterodimer that senses invading DNA directed by RNA guides. Upon target recognition, the Ago-Aga1 complex physically interacts with and activates Aga2, a transmembrane toxic effector, resulting in membrane depolarization and eventually bacterial cell death.

Akin to SPARTA systems, SPARSA systems from Geobacter sulfurreducens (GsSPARSA), Caballeronia cordobensis (CcSPARSA) and Paraburkholderia graminis (PgSPARSA) also provide immune protection via NAD+ depletion [20]. All SPARSA systems from different bacteria can protect against double-stranded (ds)DNA phage infections, while GsSPARSA and CcSPARSA can also defend against transformation of plasmids that contain a CloDF13 replication origin. For GsSPARSA, it was further demonstrated that the SIR2-APAZ protein and short Ago form a heterodimeric complex to bind RNA-DNA duplex and hydrolyse NAD(P)+. Upon RNA-guided invader DNA detection, the GsSPARSA complex becomes activated to deplete cellular NAD+, triggering bacterial cell death (Figure 2A) [20].

SPARDA systems comprise a short Ago and an effector protein containing a nuclease domain [23]. Akin to the SPARTA and SPARSA systems, the short Agos and the effector proteins of the DREN-APAZ and DUF4365-APAZ in the SPARDA system from Novosphingopyxis baekryungensis (NbaSPARDA) and Thermocrispum municipal (TmuSPARDA), respectively, also form heterodimeric complexes [23,24]. The nuclease activity of SPARDA becomes activated upon detecting invader DNA in the presence of guide RNA, leading to indiscriminate collateral cleavage of DNA and RNA and eventually resulting in cell death (Figure 2A) [23,24].

On the other hand, in the peculiar SiAgo system, the complex formed by SiAgo and SiAga1 directly interacts with the membrane effector protein SiAga2. Upon recognizing ssDNA targets, the SiAgo-Aga1 complex triggers SiAga2 activation, leading to membrane depolarization and bacterial cell death (Figure 2B) [22]. Thus, in all reported short Ago systems, short Agos form complexes with their accessory proteins to specifically recognize target DNA in the presence of guide RNA, subsequently triggering the activation of the effector proteins for immune defence (Figure 2) [19,20,22–24].

Assembly and activation of SPARTA

Several groups have reported the cryo-EM structures of SPARTA systems and proposed the assembly and activation mechanism of SPARTA [30–39]. For both MapSPARTA and CrtSPARTA systems, the SPARTA structures in different states, including a monomeric apo state, a monomeric guide RNA and target DNA (RNA-DNA) bound state, two dimeric RNA-DNA bound states (symmetric and asymmetric dimers) and a tetrameric RNA-DNA bound active state, have been reported [30,31] (Figure 3A–C). These structures in conjunction with extensive biochemical analyses unveiled a fascinating hierarchical assembly-mediated activation mechanism of SPARTA (Figure 3D). As the structural information from both systems is similar, here we use MapSPARTA as an example. The apo structures of SPARTA revealed that the short Ago and TIR-APAZ established extensive interactions, in which the APAZ domain interacts with both the MID and PIWI domain, while the TIR domain packs tightly against the MID domain. SPARTA starts as an apo-auto-inhibited monomer, where the guide-target (RNA-DNA) duplex-binding channel is occupied by an auto-inhibitory C-terminal motif (CTM) in TIR-APAZ, impeding nucleic acid binding (Figure 3D). Upon the binding of RNA-DNA, the unique configuration of the target DNA can effectively compete off the CTM from the nucleic acid-binding pocket. Furthermore, SPARTA undergoes a series of conformational changes, in particular in the MID domain. The conformational changes in the MID domain trigger the exposure of a positive-charged pocket, which is critical for mediating MID–MID dimerization. As such, SPARTA transitions from a monomer to a symmetric dimer. Charge repulsion in the TIR domains of the symmetric dimer may drive the rotation of one TIR domain to 180º, leading to the formation of an asymmetric SPARTA dimer. In the asymmetric SPARTA dimer, the interactions between TIR domains are facilitated through charge and shape complementarity (Figure 3D). Eventually, two pairs of asymmetric SPARTA dimer further assemble into a tetramer through TIR-TIR interactions. Structures of SPARTA in complex with NAD+ showed that NAD+ binds to the interface of the asymmetric TIR dimer (Figure 3D). This hierarchical assembly process ultimately resulting in the formation of an activated higher-order assembly of SPARTA with a central TIR cluster responsible for hydrolysing NAD(P)+. Further, biochemical analysis underscored the functional importance of SPARTA tetramerization in NAD(P)+ hydrolysis. Substitutions of key residues at the tetrameric interfaces led to the generation of SPARTA dimers, which can still bind NAD+ but lost catalytic activity [30]. Figure 3. Structures of SPARTA and its assembly and activation mechanism.

(A) Ribbon diagram of apo monomeric MapSPARTA (PDB: 8FEX). The TIR domain is indicated in green, APAZ in blue, MID in magenta and PIWI in yellow. (B) Ribbon diagram of MapSPARTA bound to guide RNA (red) and target DNA (blue) (PDB: 8SQL). (C) Ribbon diagrams of tetrameric MapSPARTA in complex with the guide RNA (red)-target DNA (blue) duplex (PDB: 8FFI). The four protomers of MapSPARTA are coloured in green, blue, yellow and pink, respectively. (D) Schematic diagram illustrating the oligomerization-mediated activation of SPARTA.

Assembly and activation of SPARSA

The cryo-EM structures of NAD+-bound complexes of GsSPARSA (GsSir2/pAgo) system in the absence or presence of guide and target (RNA-DNA) duplex revealed a potential mechanism of SPARSA assembly and catalysis [32,40]. The architecture of SPARSA features a heterodimer comprising a short Ago enveloped by the N-terminal Sir2 domain and the C-terminal APAZ domain. The N-terminal Sir2 domain exhibits a Z-shaped structure, connecting with the APAZ domains via a long linker. The short Ago adopts a typic MID-PIWI fold, similar to other Agos, forming a nucleic acid-binding channel with the APAZ domain (Figure 4A). An NAD+ molecule was identified in the SIR2 domain with the apo SPARSA, indicating an auto-inhibited conformation. H186 and N142 may serve as the catalytic residues, while several residues near these catalytic residues are critical for coordinating NAD+. In particular, the catalytic residues are far away from the substrate, further supporting that the apo SPARSA adopts a catalytic-inactive conformation. Figure 4. Structures of SPARSA and proposed activation mechanism.

(A) Ribbon diagram of apo monomeric GsSPARSA (PDB: 8JKZ). The SIR2 domain is indicated in blue, APAZ in green, MID in magenta and PIWI in yellow. (B) Ribbon diagram of GsSPARSA bound to guide RNA (red) and target DNA (blue) (PDB: 8JL0). (C) Schematic diagram illustrating the proposed activation mechanism of SPARSA.

Cryo-EM structures of SPARSA in complex with nucleic acids showed that guide-target (RNA-DNA) duplex is situated in the nucleic acid-binding channel formed by the short pAgo and the APAZ domain, resembling the SPARTA system (Figure 4B) [40]. Unexpectedly, RNA-DNA duplex binding triggers subtle structural rearrangements in the MID, PIWI and SIR2 domains. In particular, the active site of the SIR2 domain was still occupied by an NAD+ molecule but not the catalytic products, indicating a pre-catalytic state. Detailed structural analysis revealed that a water-mediated network contributed to the coordination and catalysis of NAD+ in the RNA-DNA bound SPARSA. Water molecules near the NAM ribose moiety of NAD+ provide possibilities for α-face nucleophilic attack on C1’. In addition, conformational changes in a loop at the catalytic site brought the catalytic residues in proximity to the NAD+ substrate, priming the NAD+ hydrolysis by SPARSA [40]. However, why the NAD+ molecule in the nucleic acid-bound SPARSA was not converted into a product remains unclear (Figure 4C). Though both SPARSA and SPARTA catalyse NAD+ hydrolysis, the catalytic domains and residues in the two systems differ [30,40]. SPARTA relies on oligomerized TIR domains for NAD+ hydrolysis, whereas SPARSA leverages a monomeric SIR2 domain to bind and hydrolyse NAD+ [30,40].

Summary and outlooks

Overall, short Agos, together with associated effector proteins, play a crucial role in prokaryotic immune defence against mobile genetic elements. Recent studies have provided structural and functional insights into their assembly and activation mechanisms. Despite being divided into different clades, short Agos and their associated effector proteins generally form heterodimeric complexes that induce cell death upon target detection and activation. SPARTA undergoes a hierarchical assembly process, transitioning from an auto-inhibited monomer to symmetric and asymmetric dimers and finally to a tetrameric active state for hydrolysing NAD(P)+. In contrast, the activation of SPARSA by nucleic acids does not seem to involve an oligomerization process. Conformational changes triggered by the nucleic acid binding may be sufficient to trigger the NAD(P)+ hydrolysis by SPARSA. The depletion of NAD(P)+ by SPARTA or SPARSA eventually leads to bacterial cell death, a common mechanism shared by many other short Ago systems.

Despite major advances in the understanding of short Ago functionality, many aspects of their functionality remain mysterious. First, how guide RNAs were generated in bacteria and where does the specificity of these RNAs towards invading DNA come from? Second, why short Ago systems like SPARTA, SPARSA and SPARDA can only be activated by RNA-DNA duplex but not RNA-RNA, DNA-DNA or DNA-RNA duplex? Additionally, structures of these systems in complex with guide RNAs alone remain to be determined. Third, as the asymmetric dimer of SPARTA can provide the catalytic site and binding site for NAD+, why is a tetrameric SPARTA required for NAD+ hydrolysis? Fourth, despite the structures of SPARSA with and without nucleic acids are available, the activation mechanism of SPARSA remains unclear. Fifth, there are many other short Ago systems with unknown functions, and structures are awaiting to be characterized. Sixth, various phage counter-defence systems that overcome prokaryotic host defence systems have been identified thus far. Is there any anti-short Ago mechanisms and if so, how do these counter defence systems function? Future biochemical, structural and cellular studies of short Ago systems will provide an in-depth understanding of these fabulous yet less-charted systems, providing a basis for reprogramming these systems into innovative therapeutics.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Statement of data availability

All the structural figures were generated using coordinates from PDB, which are available on the website of RCSB PDB.
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