# Specificity and mechanism of tRNA cleavage by the AriB Toprim nuclease of the PARIS bacterial immune system

Svetlana Belukhina, Baptiste Saudemont, Florence Depardieu, Tom Lorthios, Tinashe P. Maviza, Alexei Livenskyi, Marina Serebryakova, Maria Aleksandrova, Elizaveta Ukholkina, Nadezhda Burmistrova, et al.

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## To cite this version:

Svetlana Belukhina, Baptiste Saudemont, Florence Depardieu, Tom Lorthios, Tinashe P. Maviza, et al.. Specificity and mechanism of tRNA cleavage by the AriB Toprim nuclease of the PARIS bacterial immune system. Philosophical Transactions of the Royal Society B: Biological Sciences, 2025, 380 (1934), 10.1098/rstb.2024.0074. hal-05353021v2

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**Cite this article:** Belukhina S *et al.* 2025

Specificity and mechanism of tRNA cleavage by
the AriB Toprim nuclease of the PARIS bacterial
immune system. *Phil. Trans. R. Soc. B* **380**:
20240074.

https://doi.org/10.1098/rstb.2024.0074

Received: 3 February 2025

Accepted: 9 May 2025

One contribution of 18 to a discussion meeting
issue 'The ecology and evolution of bacterial
immune systems'.

**Subject Areas:**

molecular biology, microbiology

**Keywords:**

PARIS, bacteriophage, bacterial immunity,
nuclease, tRNA

**Authors for correspondence:**

Artem Isaev

e-mail: Artem.Isaev@skoltech.ru

David Bikard

e-mail: david.bikard@pasteur.fr

Christophe Rouillon

e-mail: christophe.rouillon@pasteur.fr

Specificity and mechanism of tRNA
cleavage by the AriB Toprim nuclease of
the PARIS bacterial immune system

Svetlana Belukhina<fnref n="1" />,†, Baptiste Saudemont<fnref n="2" />,†, Florence Depardieu<fnref n="2" />, Tom Lorthios<fnref n="2" />, Tinashe P. Maviza<fnref n="1" />, Alexei Livenskyi<fnref n="1" />,<fnref n="3" />,<fnref n="4" />, Marina Serebryakova<fnref n="3" />, Maria Aleksandrova<fnref n="1" />, Elizaveta Ukholkina<fnref n="1" />, Nadezhda Burmistrova<fnref n="3" />, Petr Sergiev<fnref n="1" />,<fnref n="3" />, Marouane Libiad<fnref n="5" />, Sarah Dubrac<fnref n="5" />, Frederic Barras<fnref n="5" />, Yuri Motorin<fnref n="6" />, Virginie Marchand<fnref n="6" />, Gregor Hagelueken<fnref n="7" />, Artem Isaev<fnref n="1" />, David Bikard<fnref n="2" /> and Christophe Rouillon<fnref n="2" />

Transfer RNA (tRNA) molecules have been recently recognized as
widespread targets of bacterial immune systems. Translation inhibition
through tRNA cleavage or modification inhibits phage propagation,
thereby protecting the bacterial population. To counteract this, some
viruses encode their own tRNA molecules, allowing infection to take
place. The AriB effector of the PARIS defence system is a Toprim
nuclease previously shown to target the *Escherichia coli* tRNA<sup>Lys(UUU)</sup>, but
not a tRNA<sup>Lys(UUU)</sup> variant encoded by bacteriophage T5. We demonstrate
here that the T5 tRNA<sup>Lys(UUU)</sup> is required but not sufficient to bypass
PARIS immunity. Combining tRNA sequencing, genetics, phage infection
and *in vitro* biochemical data, we reveal that the *E. coli* tRNA<sup>Thr(UGU)</sup> is
another prime target of AriB, and tRNA<sup>Asn(GUU)</sup> represents a secondary,
yet biologically relevant, target of the PARIS effector. Activated AriB
protein cleaves these targets *in vitro*, and the cleavage reaction is not
dependent on the presence of specific tRNA modifications. We show that
the overexpression of phage T5 tRNA<sup>Lys(UUU)</sup>, tRNA<sup>Thr(UGU)</sup> and tRNA<sup>Asn(GUU)</sup>
variants is sufficient to inhibit PARIS anti-viral defence. Finally, we propose
a model for tRNA recognition by the AriB dimer and provide molecular
details of its nuclease activity and specificity.

This article is part of the discussion meeting issue 'The ecology and
evolution of bacterial immune systems'.

**1. Introduction**

The central role of tRNAs in protein synthesis makes them prime targets for
various toxins inhibiting the growth of bacteria. To compete with neigh-
bours, some bacteria secrete bacteriocins, like colicins E5 and D, which are
known to cleave specific tRNAs within their anticodon loop [1]. Multiple
toxin-antitoxin (TA) systems also inhibit translation by targeting tRNAs [2-

<footnote n="1">¹These authors contributed equally to the study.</footnote>

<footnote n="1">¹Skolkovo Institute of Science and Technology, Moscow 121205, Russian Federation</footnote>

<footnote n="2">²Department of Microbiology, Synthetic Biology, Institut Pasteur, Université Paris Cité, CNRS UMR 3525, Paris 75015, France</footnote>

<footnote n="3">³Lomonosov Moscow State University, Moscow 119991, Russian Federation</footnote>

<footnote n="4">⁴Institute of Gene Biology, Moscow, 119334, Russian Federation</footnote>

<footnote n="5">⁵Department of Microbiology, Stress Adaptation and Metabolism Unit, Institut Pasteur, Université Paris Cité, CNRS UMR 6047, Paris 75015, France</footnote>

<footnote n="6">⁶SMP IBSLor, EpiRNA-Seq Core Facility, Université de Lorraine, Vandoeuvre-les-Nancy 54505, France</footnote>

<footnote n="7">⁷Institute of Structural Biology, Venusberg-Campus 1, University of Bonn, Bonn 53127, Germany</footnote>
4]. Although TA systems have been linked to various biological functions, seminal work [5] and more recent studies have highlighted their role as anti-phage defence systems, primarily functioning by arresting the growth of or killing the host bacteria, thereby blocking phage propagation [6]. Targeting tRNAs appears to be a common strategy among anti-phage defence systems, as highlighted by the PrrC anticodon nuclease [7], CRISPR-Cas13 [8], Retron-Eco7 [9], toxSAS-CapRel and FaRel [10,11], RemAIN [12], the PARIS immune system [13] and more [14].

Mechanisms of tRNA inactivation include their modification by nucleotidyltransferases such as MenT [15], by pyrophosphokinases such as CapRel [10], or by acetyltransferases such as TacT [16,17]. Another frequent route of tRNA inactivation involves the cleavage of the anticodon loop by proteins with a colicin D/E5 nuclease domain, by PIN-domain ribonucleases such as VapC [18,19] and MazF [20], or by HEPN nucleases such as PrrC, RloC [21] and RemN [12]. Other nuclease domains have recently been shown to be involved in the degradation of specific tRNAs in the context of anti-phage defence systems. These include the HNH nuclease domain of PtuB in the Retron-Eco7 system [9] or the Toprim domain of AriB in the PARIS system [13]. Toprim domain-containing nucleases play essential roles in DNA replication, repair and recombination [22]. This domain is also found in OLD proteins, where it is associated with an ABC ATPase domain [23]. This association has been described across several anti-phage defence systems, including the prototypical OLD protein of bacteriophage P2 [24], Gabija [25], retron elements [26], AbiL [27], MADS [28] and the PARIS [29] systems.

PARIS systems are composed of two proteins (AriA and AriB) that are sometimes fused as a single polypeptide. AriA is an ABC ATPase that assembles into a hexamer and functions as a sensor, while AriB is a tRNase that contains a Toprim domain and serves as the effector protein. In the absence of phage infection, the AriA hexamer binds to and sequesters three copies of AriB, maintaining them in an inactive state. Upon phage infection, AriA detects specific phage proteins, such as Ocr from phage T7 or Ptr1 and Ptr2 proteins from T5 [13,29,30]. These triggers bind to AriA, inducing a conformational change that leads to the release and dimerization of AriB. We recently described how the activated AriB protein cleaves the tRNA<sup>Lys(UUU)</sup> in the stem of the anticodon stem-loop between positions 40 and 41, a cleavage site not previously described for other tRNA nucleases [13]. It was proposed that viral tRNAs accumulate mutations to avoid cleavage by the host tRNases [31]. This was confirmed in the case of the T5 tRNA<sup>Lys(UUU)</sup>, which contains two mutations around the AriB cleavage site that are sufficient to provide resistance to AriB cleavage [13].

In this study, we characterize the complete set of AriB Toprim nuclease tRNA targets and describe the mechanism of cleavage in more detail. We show that in addition to tRNA<sup>Lys(UUU)</sup> AriB cleaves tRNA<sup>Thr(UGU)</sup>, and to a lower extent tRNA<sup>Asn(GUU)</sup> and tRNA<sup>Thr(CGU)</sup>. We demonstrate that cleavage of secondary tRNA targets is essential for PARIS activity, since only overexpression of the full set of phage T5 tRNA variants can rescue PARIS-induced toxicity and inhibit its anti-viral immunity. We further investigate whether base modifications found in tRNAs are required for AriB target recognition, and the RNA sequence determinants of this recognition. Finally, we employ AlphaFold 3 (AF3) to propose a model of how two catalytic sites within the AriB dimer engage the asymmetrical tRNA substrate, leading to a unique cleavage site. Altogether our results shed light on the molecular mechanism underlying tRNA cleavage by the Toprim domain of AriB, a protein domain with homologues found across many uncharacterized anti-phage defence systems.

## 2. Material and methods

### (a) tRNA extraction, sequencing and analysis

The preparation of enriched small RNA fractions from total RNA samples was performed as previously described [13].

Small RNA libraries focusing on long reads were prepared using a custom protocol. Up to 200 ng of small RNAs were dephosphorylated using Quick CIP (NEB) in 1× rCutSmart buffer at 37°C for 15 min, followed by purification with the Monarch® Spin RNA Cleanup Kit (NEB). For 3' adapter ligation, 180 ng of dephosphorylated RNA was ligated to a pre-adenylated adapter (SB104) using T4 RNA ligase 2, truncated KQ (NEB), in 1× T4 RNA ligase buffer containing 14% polyethylene glycol (PEG) 8000. The reaction was incubated at 25°C for 2 h and subsequently purified with the Monarch® Spin RNA Cleanup Kit. Reverse transcription (RT) was performed on 140 ng of 3'-ligated RNA using Maxima Reverse Transcriptase (Thermo Fisher Scientific) in 1× RT buffer with ribonuclease (RNase) inhibitors and 1.3 µM RT primer (SB103). The reaction was conducted under the following conditions: 65°C for 5 min, 60°C for 30 min, 65°C for 25 min and 80°C for 5 min. RNase H and RNase A treatments were performed post RT, followed by purification with the Monarch® Spin RNA Cleanup Kit. For 5' adapter ligation, the complementary DNA (cDNA) was ligated to a 5' adapter (SB077) using Thermostable 5'App DNA/RNA Ligase (NEB) in 1× NEB1 buffer with 5 mM MnCl₂. The reaction was incubated overnight at 65°C and purified twice with the Monarch® Spin RNA Cleanup Kit. Library amplification was conducted using indexed primers (SB085 and SB071 or SB072) on 2 ng of cDNA with Q5® High-Fidelity DNA Polymerase (NEB) in 1× Q5 buffer supplemented with 1× GC-rich enhancer. PCR conditions included 25 cycles with an annealing temperature of 60°C. A quarter of the amplified library was resolved on a 10% denaturing polyacrylamide gel electrophoresis (PAGE) with 7M urea. Fragments >85 nt were excised, eluted by soaking overnight (or for 2 h at room temperature), and purified with Monarch® Spin RNA Cleanup Kit columns. Multiplexed libraries were sequenced on an Illumina NextSeq 2000 platform in a 150 bp paired-end configuration.

To map 5' ends of small tRNA fragments, approximately 100 ng of purified tRNAs from *Escherichia coli* (with or without activated PARIS) or *in vitro* reactions with purified AriB were end-repaired as described previously [32]. RNA was purified using the RNeasy MinElute Cleanup Kit (Qiagen) according to the manufacturer's protocol, with a modified RNA binding step that included 675 µl of 96% ethanol. Purified RNA fragments were eluted in 10 µl of nuclease-free water. Library preparation was performed using the commercial kit NEBNext® Small RNA Library Prep Set for Illumina (no. E7330S, NEB, USA) following
the manufacturer's instructions. DNA libraries were quantified using a Qubit 2.0 fluorometer (Invitrogen, USA) and assessed for quality using a High Sensitivity DNA chip on an Agilent Bioanalyzer 2100. Libraries were multiplexed and sequenced on an Illumina NextSeq 2000 platform using 50 bp single-end read mode.

Reads were mapped to a list of *E. coli* tRNAs and rRNAs, excluding any redundant sequences. The alignment algorithm used was Bowtie2 with default parameters, and only uniquely mapped reads were kept for the scatter plots.

### (b) AriB purification

To produce activated AriB, PARIS (AriA-AriB-Strep) from pFR85 and non-tagged Ocr from pBAD were expressed in separate cultures as previously described [13]. Cell cultures were centrifuged and pellets resuspended in buffer A (100 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA) containing protease inhibitor cocktail (Abcam, ab271306). Cells were lysed by sonication on ice and cell debris was removed by centrifugation (15 000g, 20 min, 4°C). Cleared filtered lysates of PARIS- and Ocr-expressing cells were mixed and incubated for 1 h at room temperature before loading on a 5 ml StrepTrap XT affinity column (Cytiva) equilibrated in buffer A. Activated AriB-Strep was eluted with buffer BXT (IBA Lifesciences). Further purification was done with size exclusion chromatography performed on a Superdex 200 Increase 10/300 column (Cytiva) equilibrated with buffer B (20 mM Tris pH 8.0, 250 mM NaCl, 1 mM dithiothreitol (DTT)). Fractions of interest were combined and concentrated (Amicon Ultra Centrifugal Filter, 30 kDa molecular weight cut-off).

### (c) DNA template production for toeprint

First, we designed a DNA matrix for T7 *in vitro* transcription that will encode an ORF, utilizing all known *E. coli* tRNA species (*RST3_all_tRNAs*; electronic supplementary material, table S1). To generate this dsDNA matrix we used two partially overlapping oligos encoding the ORF and carrying T7 binding site. Oligos were annealed with gradual temperature decrease from 98 to 25°C at 2°C every 2 min. To synthesize the missing second strand, DNA Polymerase I, Large (Klenow) Fragment (NEB) was used according to the manufacturer's instructions. After the reaction, DNA templates were purified from excess dNTPs and polymerase with the Monarch® PCR & DNA Cleanup Kit (NEB).

### (d) *In vitro* translation and toeprinting analysis

The generated DNA templates were amplified using Q5® High-Fidelity 2X Master Mix (NEB) with T7F (TAATACgACTCACTA-TAgg) and NV1 (GGTTATAATGAATTTTGCTTATTAAC) primers. The templates were validated for *in vitro* transcription using the MEGAscript® Kit (Thermofisher Scientific) according to the manufacturer's instructions, supplemented with FAM-11-UTP (0.25 mM). To assess RNA integrity of T7 transcript(s), RNA samples were subjected to 6% denaturing polyacrylamide-urea gel electrophoresis, followed by scanning using Typhoon™ FLA 9500. Toeprinting was performed using the PURExpress *In Vitro* Protein Synthesis Kit (NEB no. E6800) and fluorescently labelled probes [33]. The mixture, containing 2 µl of solution A, 1 µl of solution B, 0.5 µl of 20 µM FAM-labelled NV1 primer (5'-/FAM-/GGTTATAATGAATTTTGCTTATTAAC-3', bearing a fluorescein at the 5'-end) and 0.2 µl of 40 U µl⁻¹ Ribolock (Thermofisher Scientific), was treated with either 0.5 µl of 0.6 µg ml⁻¹ AriB or storage buffer (TAKM₇ buffer: 50 mM Tris-HCl pH 7.5, 70 mM NH₄Cl, 30 mM KCl and 7 mM MgCl₂) for 20 min at 37°C. Thiostrepton (Ths) (50 µM) and dimethyl sulfoxide (DMSO) 1% were used as a control to denote the inhibitory signal at initiation stage. The reactions were started when a DNA template (10 ng) of interest was added to the mixture, followed by an additional incubation for 30 min at 37°C. A 1 µl master mix containing 0.1 µl AMV reverse transcriptase (Roche), 0.2 µl of 5× AMV buffer (Roche), 0.5 µl of 4 mM dNTP mix (Thermofisher Scientific) and 0.2 µl of ultra-pure water was added to the reaction(s) for cDNA synthesis. The AMV reverse transcription reaction was conducted for 15 min at 37°C. The reaction(s) were then terminated using 1 µl of 10 M NaOH, with subsequent incubation for 15 min at 37°C, followed by complete neutralization by adding 1 µl of 12 M HCl. The cDNA product was purified using the QIAquick PCR Purification Kit (Qiagen). Fluorescently labelled cDNA products were analysed by capillary gel electrophoresis using a Nanophore-05 Genetic Analyzer (Syntol, Russia). The collected data was processed and visualized using GeneMarker® software (SoftGenetics). The abundance of cDNA fragments scored was denoted by area under the graph which reflects the amount of stalled ribosomes. The ribosome position was deduced by using the classical toeprinting principle. The 3' end of synthesized cDNA product is separated by approximately 13 or 14 nt from the first base of the A-site when reverse transcriptase encounters stalled ribosomes. All experiments were conducted reproducibly at least three times.

### (e) *Escherichia coli* mutants deficient for tRNA modifications

Strains harbouring *ΔmnmA*, *ΔtruA*, *ΔtruB* or *ΔtcdA* were obtained from the Keio collection. Each deletion was transferred individually to strain MG1655 (FBE051) by P1 transduction, and the kanamycin resistance selection marker was then removed using the pE-FLP (Amp⁶) plasmid, able to recombine FRP (Flipase Recognition Target) sites flanking the backbone. pE-FLP was then cured through serial restreaks on LB (Luria Bertani) plates, leading to strains FBE939 (*ΔmnmA*) [34], FD6 (*ΔtruA*), FD7 (*ΔtruB*) and FD8 (*ΔtcdA*). The integration and then each deletion on the chromosome were checked by PCR with primers followed by Sanger sequencing.

Each strain harbouring the various deletions was transformed, by heat shock at 42°C following standard protocols, with pFR85 (Ptet *ariA-ariB*) or control plasmid pFR66 (Ptet sfGFP) and pFD250 (PPhlF *ocr*) carrying *ocr* under the control of an
inducible 2,4-diacetylphloroglucinol (DAPG) PhlF promoter. Then *E. coli* cells carrying these plasmids were grown in LB broth with kanamycin (50 µg ml⁻¹) for pFR85 and pFR66 and chloramphenicol (20 µg ml⁻¹) for pFD250 (PPhlF ocr) overnight at 37°C, followed by serial dilutions, and spotted on LB agar containing kanamycin and chloramphenicol with induction of Ocr or not by DAPG 50 µM. Strains, primers and plasmids are listed in electronic supplementary material, table S1.

### (f) In vitro transcript production and purification

Oligonucleotides containing various tRNA sequences preceded by a 5' extended T7 promoter sequence (CGATTGAGGCCGGTAATACGACTCACTATA) were obtained from IDT or Merk (oligos: electronic supplementary material, table S1). A 20-cycle PCR was run on 3 ng of template oligo with a forward primer corresponding to the 5' extended T7 promoter sequence and a template-specific reverse primer antisense to 30 nt of the 3' end of the template oligo in an NEB Q5® High-Fidelity reaction mix. Products were purified with a Macherey-Nagel PCR cleanup column, eluted in 30 µl.

PCR product (0.8–1 µg) was then used as a template for *in vitro* transcription (IVT) with a HiScribe® T7 High Yield RNA Synthesis Kit. DTT (5 mM), 10 mM of each NTP and 2 µl of T7 RNA Polymerase Mix in 20 µl were reacted overnight at 37°C. The IVT product was either PAGE-purified or purified using an NEB Monarch® RNA Cleanup column, following DNase I treatment, and eluted in 20 µl RNase-free water.

### (g) In vitro AriB nuclease activity

*In vitro* activity on total enriched small RNA was determined as described in [13]. The activity on *in vitro* transcribed tRNA was measured in 20 µl reaction buffer (20 mM Tris pH 8.0, 200 mM NaCl) containing 100 nM (2 pmol) purified tRNA and 25 nM pure AriB dimer. MgSO₄ was added to a final concentration of 1 mM, except for the experiment on metal-dependency, where the final concentration was 0.5 mM, alongside other metals (MnCl₂, CaCl₂, CoCl₂, CuSO₄ and NiCl₂). The mixture was incubated at 37°C for 20 min, unless otherwise specified, and the reaction was stopped with an equal volume of 100% formamide. The samples were incubated for 5 min at 92°C before loading 20 µl (1 pmol) on a preheated 12% denaturing PAGE gel containing 7 M urea. After running the gel at 25 W constantly for about an hour, the gel was stained with SYBR™ Gold (Invitrogen S11494) for 10 min before visualization on a Bio-Rad Chemidoc MP Imaging System.

### (h) Poly(A) tailing of AriB cleavage products

In 10 µl 1× poly(A) reaction buffer, 2 pmol (200 nM final concentration) of purified (NEB Monarch® RNA Cleanup column) AriB-cleaved tRNA<sup>Lys(UUU)</sup> was incubated with 1 mM ATP and 2.5 units of *E. coli* poly(A) polymerase (NEB M0276) for 30 min at 37°C. The reaction was stopped with an equal volume of 100% formamide, and incubated for 5 min at 92°C before loading 1 pmol on a preheated 12% denaturing PAGE gel alongside 1 pmol of uncut and cut controls.

### (i) Phage infection: efficiency of plaquing assay

The activity of PARIS or PARIS mutant pFD340 (Ptet *ariA* *ariB* K60A/K64A) was measured by performing efficiency of plaquing (EOP) assays with different phages. *Escherichia coli* BW25113 carrying the control plasmid pFR66 (sfGFP) or the wild-type (WT) PARIS system (pFR85) with pBAD vector encoding tRNAs was grown overnight in LB with kanamycin (50 µg ml⁻¹) and ampicillin (100 µg ml⁻¹). *Escherichia coli* K-12 MG1655 carrying the double mutant pFD340 (Ptet *ariA* *ariB* K60A/K64A), the control plasmid pFR66 (sfGFP) or pFR85 (Ptet *ariA* *ariB* or WT PARIS system) was grown overnight in LB supplemented with 50 µg ml⁻¹ kanamycin. Bacterial lawns were prepared by mixing 100 µl of a stationary culture with 5 ml of LB + 0.5% agar, and the mixture was poured onto a Petri dish containing LB with kanamycin (50 µg ml⁻¹) for pFR85 (Ptet *ariA* *ariB* or WT PARIS system), pFR66 (sfGFP) and pFD340 (Ptet *ariA* *ariB* K60A/K64A) and ampicillin (100 µg ml⁻¹) in the presence of pBAD vector derivatives and supplemented with 1 mM CaCl₂. Induction of tRNA genes was achieved by addition in the top agar of the indicated amount of L-arabinose (L-Ara). Tenfold serial dilutions of a high-titer stock of phages were spotted on each plate and incubated overnight at 37°C, or 5 h for T7 phage. Plaque assays were performed in at least two independent replicates.

### (j) Solid medium toxicity assay

PARIS toxicity in the presence of T7 Ocr was measured using a spot-test assay. PARIS⁺ (pFR85) and PARIS⁻ (pFR66) strains carrying the pFD250 (PPhlF Ocr) with/without pBAD vector carrying tRNAs from T5 phage were grown overnight in 10 ml of LB + kanamycin (50 µg ml⁻¹) + ampicillin (100 µg ml⁻¹) + chloramphenicol (34 µg ml⁻¹) at 37°C. Stationary cultures were diluted to OD₆₀₀ 0.6 and plated on LB (figure 3A) or minimal medium (M9 with 5% v/v LB) (figure 2A) agar plates by serial tenfold dilution; plates were supplemented with various L-Ara percentages (0.02–2%) or/and DAPG (30–50 µM).

### (k) Mutant (K60A and K64A)

The double mutation K60A and K64A in *ariB* was synthesized with primers F747 and F748 (electronic supplementary material, table S1) and introduced on the pFR85 plasmid carrying *ariA* and *ariB* genes with the native promoter and an inducible Ptet
promoter upstream through two PCRs with primers F747/LC327 and TG99/F748 (electronic supplementary material, table S1) of the whole plasmid, followed by Gibson assembly [35]. The resulting plasmid pFD340 (*ariA ariB* K60A/K64A) was introduced into competent *E. coli* K-12 MG1655 cells by heat shock at 42°C, following standard protocols, and plated on LB agar supplemented with 50 µg ml⁻¹ kanamycin. The construction was verified using Sanger sequencing. Activity of this mutant was tested by performing EOP assay as described above.

### (I) Structure by AlphaFold 3 and alignment with M5 nuclease

Two sequences of AriB monomer and the sequence of *E. coli* tRNA<sup>Lys(UUU)</sup> were loaded on the AF3 server. The predicted template modelling (pTM) score of 0.92 means the overall predicted fold for the complex might be similar to the true structure [36], and the interface predicted template modelling (ipTM) score of 0.82 suggests a high-confidence prediction [37]. The 'align' command in PyMOL was used to superpose the M5 nuclease (PDB 6z2b) onto AriB. Specifically, chain B of the M5 nuclease was aligned to a selection comprising residues 19–43, 48–51, 80–87, 109–111 and 113–115 of AriB. This led to a root mean square deviation (r.m.s.d.) of 0.47 Å over 87 pairs of atoms.

### (m) Mass spectrometry analysis of tRNA<sup>Lys(UUU)</sup> fragments

Five micrograms of *in vitro* transcribed *E. coli* tRNA<sup>Lys(UUU)</sup> was mixed with 1 µl AriB or AriB E26A (16 mg ml⁻¹) in the reaction buffer (25 mM Tris-HCl, 35 mM NH₄Cl, 15 mM KCl, 5 mM MgCl₂, 0.5 mM DTT, pH 7.5). The reaction mixture was incubated at 37°C for 30 min. Purification of tRNA and fragments from reaction was performed with Lira reagent (LR-100, Biolabmix, Russia). Afterwards, re-extracted tRNA was mixed with 20 U RNase T1 (Thermo Fisher Scientific, USA) in 30 mM ammonium acetate and incubated at 37°C for 30 min. A part of this reaction was then mixed with 2,5-dihydroxybenzoic acid solution (40 mg ml⁻¹ in 30% acetonitrile, 0.5% trifluoroacetic acid) and analysed by mass spectrometry using an UltraflexXetreme MALDI-TOF/TOF spectrometer (Bruker Daltonik, Germany) equipped with a Nd laser. The average MH⁺ molecular ions of RNase T1-treated tRNA were measured in linear mode; the accuracy of average mass peak measurement was within 1 Da.

## 3. Results

### (a) Landscape of tRNA cleavage by AriB

The tRNA<sup>Lys(UUU)</sup> was previously identified as a target of PARIS based on the observation that the T5 tRNA<sup>Lys(UUU)</sup> could rescue the infection of a deletion variant of T5 (T5<sub>123</sub>) in which 11 of the 24 tRNAs were lost (electronic supplementary material, figure S2A). Since T5<sub>123</sub> still carries 13 tRNA genes, we could not exclude that AriB also cleaves additional *E. coli* tRNAs, and that other phage tRNAs contribute to countering PARIS immunity. To test this hypothesis, we expressed the T5 tRNA<sup>Lys(UUU)</sup> in the presence of PARIS during infection by T5<sub>Mos</sub>, another T5 deletion variant that has lost a slightly different set of 17 tRNA genes [13] (electronic supplementary material, figure S2A). Although T5 tRNA<sup>Lys(UUU)</sup> expression completely rescued T5<sub>123</sub> infectivity, it was not sufficient in the case of T5<sub>Mos</sub> (figure 1A). Thus, tRNA<sup>Lys(UUU)</sup> from T5 is necessary, but not sufficient to counteract the effect of AriB activation, suggesting the existence of additional tRNA targets.

To determine the comprehensive set of AriB targets, we aimed to sequence tRNAs extracted 30 min following the induction of T7 Ocr in *E. coli* cells expressing PARIS or a GFP control (figure 1B–D; electronic supplementary material, figure S1A). We first implemented a custom protocol that yielded a high number of reads covering more than 80% of the tRNA length. These reads served as a proxy to calculate the abundance of full-length, functional tRNAs. This analysis revealed a depletion of tRNA<sup>Lys(UUU)</sup>, tRNA<sup>Thr(UGU)</sup>, tRNA<sup>Asn(GUU)</sup> and tRNA<sup>Thr(CGU)</sup> following the induction of PARIS and its trigger (figure 1B). We were, however, unable to clearly identify tRNA cleavage products in this dataset. tRNAs contain numerous modified bases that can cause the reverse transcriptase enzyme to stall, resulting in shorter cDNAs compared with their templates [38,39]. Our custom protocol was adding sequencing adapters to cDNAs, resulting in high-coverage sequencing of these short cDNAs which can obfuscate cleavage products. To address this, we used a commercial kit that ligates sequencing adapters to RNA ends prior to RT, ensuring PCR amplification is limited to fully reverse-transcribed products. Using these data, analysis of 5' ends within the anticodon stem-loop identified probable cleavage products for tRNA<sup>Lys(UUU)</sup>, tRNA<sup>Thr(UGU)</sup>, tRNA<sup>Asn(GUU)</sup>, tRNA<sup>Thr(CGU)</sup>, tRNA<sup>His(GUG)</sup> and tRNA<sup>Pro(UGG)</sup> (figure 1C). Some of the tRNAs detected with this method could be minor targets of AriB that are not cleaved efficiently enough to show a depletion signal from the pool of full-length tRNAs. Notably, a cleavage between position 40 and 41 could be detected for most of these tRNAs (electronic supplementary material, figure S1B), as previously described for the tRNA<sup>Lys(UUU)</sup>. Some of these cleavage products could also be indirect consequences of translation arrest after AriB activation. To further validate the targets of AriB we performed an *in vitro* cleavage assay in which *E. coli* tRNAs were incubated with activated AriB protein (electronic supplementary material, figure S1A). Sequencing of this sample enabled the detection of cleavage events in the tRNA<sup>Lys(UUU)</sup>, tRNA<sup>Thr(UGU)</sup> and tRNA<sup>Asn(GUU)</sup> (figure 1D). Altogether, the main targets of AriB appear to be tRNA<sup>Lys(UUU)</sup>, tRNA<sup>Thr(UGU)</sup> and tRNA<sup>Asn(GUU)</sup>.

To test whether tRNA cleavage by AriB would lead to ribosomal stalling at the expected codons, we performed toeprinting analysis. In this assay, mRNAs undergoing *in vitro* translation are subjected to reverse transcription with fluorescein-labelled primer. The reverse transcriptase is blocked by stalled ribosomes, mapping their position (electronic supplementary material, figure S1C). To unambiguously identify codons affected by AriB tRNA cleavage, we designed an mRNA template that contains
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

**Figure 1.** Landscape of AriB-mediated tRNA cleavages. (A) Plaque assays were conducted using cells without (green fluorescent protein (GFP) control) or with PARIS in the presence of empty vector (pBAD) or pFD287 (paraBAD T5 tRNA<sup>Lys(UUU)</sup>) against T5<sub>WT</sub>, T5<sub>Mos</sub> and T5<sub>123</sub>. Overexpression of T5 tRNA<sup>Lys(UUU)</sup> in PARIS cells from pFD287 plasmid, with the tRNA<sup>Lys(UUU)</sup> of phage T5 under the control of the araBAD promoter inducible by L-Ara (0.2%), rescues infection by phage T5<sub>123</sub> but not by T5<sub>Mos</sub>. (B–D) Scatter plots of normalized tRNA abundances. Axes represent the normalized abundance of tRNAs in the respective samples. Blue dots indicate the majority of tRNAs, while red dots highlight specific tRNAs of interest: Lys(UUU), Thr(UGU), Thr(CGU) and Asn(GUU). The dashed grey line shows the linear regression fit in log–log space. (B) tRNAs with mapped reads ≥65 nt from GFP control and PARIS (*in vivo* cultures), sequenced using a custom protocol (see §2). (C) Small tRNAs (<45 nt) with 5' ends mapped to the anticodon stem–loop, from GFP control and PARIS (*in vitro* cultures), sequenced using a commercial kit. (D) Small tRNAs (<45 nt) with 5' ends mapped to the anticodon stem–loop, from untreated GFP control and *in vitro* AriB cleavage samples, sequenced using a commercial kit. (E,F) Frequency of ribosome pausing on specific codons, estimated by toeprinting analysis with substrates *RST3_all_tRNAs* (E) and *RST3_all_tRNAs* (*K5A*) (F) mRNA templates translated in the presence of AriB wild-type (WT) or E26A Toprim mutant. Data are presented as relative fluorescence units (rfu). Sequences of mRNA templates are provided at the top. 'Ths' represents a control reaction with thiostrepton, which stalls ribosomes at the start codon. TAKM7 is a standard control buffer for *in vitro* translation. Signals from the start (AUG), lysine (AAA) and threonine (ACA) codons were calculated as the mean area under the curve from the capillary electropherograms carried out in triplicates; representative electropherograms are presented in electronic supplementary material, figure S1D,E. DMSO, dimethyl sulfoxide.

a codon for each known *E. coli* tRNA species [38]. Toeprinting revealed that presence of the AriB WT, but not AriB E26A Toprim mutant, resulted in the accumulation of ribosomes stalled at the lysine AAA codon (figure 1E; electronic supplementary material, figure S1D). To identify secondary AriB targets, we mutated the lysine codon in the mRNA to an alanine codon and repeated toeprinting, which revealed ribosome pausing at the downstream threonine ACA codon, recognized by tRNA<sup>Thr(UGU)</sup>,
although milder compared with the lysine codon (figure 1F; electronic supplementary material, figure S1E). Mutating both AAA and ACA tRNA codons to alanine codons did not reveal significant ribosome pausing at other sites. These in vitro results confirm that AriB cleavage of tRNA<sup>Lys(UUU)</sup> and tRNA<sup>Thr(UGU)</sup> blocks translation at the expected codons. Cleavage of the tRNA<sup>Asn(GUU)</sup> was likely not efficient enough to substantially stall ribosomes in this assay.

## (b) Multiple T5 tRNAs are needed to rescue AriB toxicity and inhibit PARIS immunity

We decided to take the validated targets tRNA<sup>Lys(UUU)</sup> and tRNA<sup>Thr(UGU)</sup>, as well as the tRNA<sup>Asn(GUU)</sup> detected by sequencing, to test whether co-expression of T5 homologues of those tRNAs is sufficient to inhibit AriB toxicity. To do so we used cells expressing Ocr (figure 2A, top) or PARIS and Ocr (figure 2A, bottom) and induced T5 tRNA expression at different arabinose concentrations. When Ocr was induced, cells expressing PARIS died as a result of AriB activation (figure 2A, right). Expression of tRNA<sup>Lys(UUU)</sup> alone did not rescue PARIS toxicity, while co-expression with tRNA<sup>Thr(UGU)</sup> increased cell survival. Growth could, however, be almost completely rescued by the co-expression (0.2% arabinose induction) of the three T5 tRNA<sup>Lys(UUU)</sup>/tRNA<sup>Thr(UGU)</sup>/tRNA<sup>Asn(GUU)</sup> (figure 2A). In conditions of enhanced expression (2% arabinose induction), co-expression of the two tRNA<sup>Lys(UUU)</sup>/tRNA<sup>Thr(UGU)</sup> was sufficient to restore cell growth (figure 2A).

We then tested whether the expression of one or several phage T5 tRNAs would rescue the infection of phages susceptible to the PARIS defence system. We performed an EOP assay with the same phages in cells expressing T5 tRNA<sup>Lys(UUU)</sup>, T5 tRNA<sup>Lys(UUU)</sup>/tRNA<sup>Thr(UGU)</sup>, T5 tRNA<sup>Lys(UUU)</sup>/tRNA<sup>Asn(GUU)</sup> or T5 tRNA<sup>Lys(UUU)</sup>/tRNA<sup>Thr(UGU)</sup>/tRNA<sup>Asn(GUU)</sup> (figure 2B; electronic supplementary material, figure S2B). As previously observed, infectivity of T5<sub>123</sub> was rescued by expressing only T5 tRNA<sup>Lys(UUU)</sup>, while infectivity of T5<sub>Mos</sub> was rescued only in conditions of overexpression the three tRNAs even though it encodes its own copy of tRNA<sup>Thr(UGU)</sup>. This suggests that the expression of tRNA<sup>Thr(UGU)</sup> from the T5<sub>Mos</sub> genome is not sufficient to rescue PARIS activation. We further noticed that T5 relatives, phages Bas33 and Bas34, which are naturally sensitive to PARIS defence, encode either tRNA<sup>Thr(UGU)</sup> or tRNA<sup>Lys(UUU)</sup> but not both (electronic supplementary material, figure 2B; figure S2A). Expression of tRNA<sup>Lys(UUU)</sup> was sufficient to rescue Bas33 infection, while rescue of Bas34 required all three tRNAs. Here, expression of the tRNA<sup>Asn(GUU)</sup> from the Bas34 genome is likely not sufficient to bypass PARIS activation. Alternatively, we cannot exclude that this tRNA is cleaved by AriB. We next showed that phage T4, which encodes tRNA<sup>Thr(UGU)</sup>, can be rescued by expression of T5 tRNA<sup>Lys(UUU)</sup>. Finally, infection of the T7 phage, which carries no tRNAs, could be rescued by the co-expression of T5 tRNA<sup>Lys(UUU)</sup>/tRNA<sup>Thr(UGU)</sup> or the three T5 tRNAs (tRNA<sup>Lys(UUU)</sup>/tRNA<sup>Thr(UGU)</sup>/tRNA<sup>Asn(GUU)</sup> (figure 2B).

Together, these results demonstrate that various viral tRNAs contribute to the inhibition of PARIS immunity, and confirm previous observations of AriB tRNA specificity, but raise additional questions regarding the mechanism of escape. It is somewhat surprising that the T5 tRNA<sup>Asn(GUU)</sup> was required to rescue infection of T5<sub>Mos</sub>, but not T4 and T7, lacking this tRNA. At the same time, expression of tRNA<sup>Thr(UGU)</sup> from plasmid was required to rescue T5<sub>Mos</sub> infection, and expression of T5 tRNA<sup>Asn(GUU)</sup> was required to rescue Bas34 infection, despite those tRNAs being encoded in the respective phage genomes. These differences could highlight the varied sensitivity of phages to the depletion of certain tRNAs. It is also possible that in addition to rescuing translation, some viral tRNAs act as inhibitors of AriB when overexpressed, maybe through competition with the *E. coli* tRNAs.

## (c) tRNA modifications are not essential for specific AriB cleavage

We then investigated the importance of tRNA modifications for AriB recognition and cleavage. In *E. coli*, there are 43 known tRNA modifications, some of which are required for cleavage by anticodon nucleases. PrrC, which cleaves the tRNA<sup>Lys(UUU)</sup> of *E. coli* between positions U35 and U36 requires cyclic threonylcarbamoyl adenosine (ct6A) at position 37 [40]. The bacterial toxin colicin E5 targets tRNAs with queuosine modification in the wobble position of their anticodon and colicin D preferentially targets modified tRNA<sup>Arg(CGN)</sup> [1]. In *E. coli* tRNA<sup>Lys(UUU)</sup>, 10 nucleotides are modified with seven different types of modifications [41]. Using Modomics [42], we revealed that *E. coli* tRNAs Lys(UUU), Thr(UGU), Asn(GUU) and Thr(CGU) contain 13 modifications distributed on similar nucleotides (electronic supplementary material, figure S3A).

To assess the importance of these modifications for PARIS activity, we used *E. coli* strains that cannot produce certain modifications, including pseudouridines Y39 (ΔtruA), Y55 (ΔtruB), ct6A37 (ΔtcdA) [43] and mnm<sup>5</sup>s<sup>2</sup>U34 in the wobble position (ΔmnmA) [34]. For all mutants tested, we found that PARIS activation triggers cell death at the same level as the WT strain (figure 3A).

We further confirmed that activated AriB can cleave tRNAs extracted from four *E. coli* mutant strains (ΔtruA, ΔtruB, ΔtcdA and ΔmnmA) *in vitro* (figure 3B). Those data suggest that AriB, by contrast to PrrC- or tRNA-targeting colicins, does not require base modifications to cleave its target. To further validate this, we tested the activity of purified AriB on an *in vitro* transcribed tRNA<sup>Lys(UUU)</sup>, lacking all modifications. In accordance with recently published results [30], we were able to see a strong cleavage of the transcript at the expected nucleotide position (electronic supplementary material, figure S3B). By contrast, AriB shows no cleavage of the control *E. coli* tRNA<sup>Phe(AAAA)</sup> (electronic supplementary material, figure S3B). Thus, base modifications are not required for AriB cleavage *in vitro*, but it remains possible that base modifications contribute to the interaction of AriB with tRNA targets *in vivo*.

## (d) Sequence specificity determinants of AriB tRNase

To gain further insights on the tRNA specificity, we challenged AriB with a larger set of *in vitro* transcribed tRNAs. The tRNA<sup>Thr(UGU)</sup> substrate was efficiently cleaved by AriB, although not as efficiently as tRNA<sup>Lys(UUU)</sup> (figure 3C). The tRNA<sup>Asn(GUU)</sup>
<figure><img src="image_10.png" /><figcaption>**Figure 2.** Expression of viral tRNAs reduces AriB toxicity and restores phage infectivity. (A) Phage T5 tRNA<sup>Lys(UUU)</sup>/tRNA<sup>Thr(UGU)</sup>/tRNA<sup>Asn(GUU)</sup> expression can rescue PARIS toxicity triggered by the expression of the phage T7 Ocr from 2,4-diacetylphloroglucinol (DAPG)-inducible PPhIF promoter. Top: without the PARIS system. Bottom: with the PARIS system. (B) Heatmap of PARIS defence against a panel of phages. Colour intensity indicates plaque-forming units (PFUs) on a log<sub>10</sub> scale. The panel below shows whether phages encode their own variant of the tested tRNAs. Representative plates used to build the heatmap as well as error bars are shown in electronic supplementary material, figure S2B,C.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 3.** AriB does not require tRNA base modifications for cleavage. (A) AriB toxicity was triggered with 2,4-diacetylphloroglucinol (DAPG, 50 µM) to induce the expression of T7 Ocr under the DAPG-inducible PPhIF promoter in *Escherichia coli* (Ec) strains harbouring different deletions of genes implicated in base modifications with either a green fluorescent protein (GFP) control vector (A) or the PARIS system (B). *In vitro* AriB cleavage assay on tRNAs extracted from the four deletion strains or the wild-type (WT) strain. (C) Activity of AriB on specific *in vitro* transcripts corresponding to *E. coli* tRNAs. (D) Activity of AriB on *E. coli* (Eco) and T5 tRNA<sup>Lys(UUU)</sup> and mutants; point mutations are shown in electronic supplementary material, figure S3D.</figcaption></figure>

demonstrated significantly reduced cleavage, whereas tRNA<sup>Thr(CGU)</sup> cleavage was barely visible on the gel, and cleavage of other control tRNAs (i.e. tRNA<sup>Gln(UUG)</sup> and tRNA<sup>Met(CAU)</sup>) was not detected (figure 3C). Interestingly, the sequence UAAU present near the tRNA<sup>Lys(UUU)</sup> cleavage site U<sub>36</sub>AAUC|A<sub>41</sub> (cleavage marked with |) is conserved in the other major target tRNA<sup>Thr(UGU)</sup> and in both minor targets tRNA<sup>Asn(GUU)</sup> and tRNA<sup>Thr(CGU)</sup> (electronic supplementary material, figure S3C). Regarding the cleavage site, whereas tRNA<sup>Thr(UGU)</sup> has the same sequence as tRNA<sup>Lys(UUU)</sup>, the tRNA<sup>Asn(GUU)</sup> has a substitution at position 41 (U<sub>36</sub>AAUC|C<sub>41</sub>) and tRNA<sup>Thr(CGU)</sup> has substitutions on both nucleotides 40 and 41 at the cleavage site U<sub>36</sub>AAUG|C<sub>41</sub> (electronic supplementary
<figure><img src="image_12.png" /><figcaption>**Figure 4.** Predicted structure and mechanism of AriB. (A) Overall structure of AriB dimer bound to *Escherichia coli* tRNA<sup>Lys(UUU)</sup> generated with AlphaFold 3 and showing the monomers AriB and AriB' in light blue and light green. The cleavage site is marked by a magenta sphere. (B) Left: view of the electrostatic surface of the AriB dimer bound to the *E. coli* tRNA<sup>Lys(UUU)</sup>, showing the two lysines K60 and K64 stabilizing the anticodon loop on the AriB monomer with the active catalytic site. Right: mutations predicted to stabilize the anticodon loop (K60A, K64A) prevent PARIS-mediated defence. Efficiency of plaquing of phage T7 on *E. coli* MG1655 carrying the wild-type (wt) or mutated PARIS system. (C) Superposition of the M5 active site with the AriB active site in complex with the *E. coli* tRNA<sup>Lys(UUU)</sup>. (D) Active catalytic site of AriB represented with two Mg<sup>2+</sup> ions modelled. (E) Incubation of AriB cleavage products with poly(A) polymerase run on a 12% denaturing gel: lane L, ssRNA ladder; lane 1, 100 nM transcript tRNA<sup>Lys(UUU)</sup>; lane 2, same as lane 1, + 25 nM AriB dimer + 1 mM Mg<sup>2+</sup>; lane 3, same as lane 2, with poly(A) polymerase treatment post cleavage.</figcaption></figure>

material, figure S3C). We previously noticed that T5 tRNA<sup>Lys(UUU)</sup>, which is resistant to cleavage by AriB [13], had two substitu-
tions we called Mut1 (U39→A) and Mut2 (A41→C). Here, using *in vitro* transcribed tRNA we confirmed that the T5 tRNA<sup>Lys(UUU)</sup>
is not cleaved by AriB (figure 3D). The mutation Mut1 (U39→A) introduced into the *E. coli* tRNA<sup>Lys(UUU)</sup> transcript completely
abolishes AriB cleavage, whereas Mut2 (A41→C) substantially inhibits it (figure 3D; electronic supplementary material, figure
S3D). To investigate whether the recognition of the deduced AriB site is sufficient to promote tRNA cleavage, we reintroduced
the *E. coli* cleavage site into the T5 tRNA<sup>Lys(UUU)</sup> (U36AAACC41 → U36AAUCA41), but this new hybrid tRNA was not cleaved
by AriB (figure 3D). This suggests that, in addition to recognition of the cleavage site, AriB forms additional base-specific
interactions with tRNAs at positions distal to the anticodon loop, which differ between T5 and *E. coli* tRNA<sup>Lys(UUU)</sup>.

(e) The two catalytic sites of the AriB dimer interact with distinct tRNA elements

To better understand how AriB distinguishes between target and non-target tRNAs and why the AriB dimer produces a single nick at the cleavage site, we predicted the structure of the AriB dimer in complex with the *E. coli* tRNA<sup>Lys(UUU)</sup> using AF3 [44]. This resulted in a structural prediction with a high degree of overall confidence (pTM 0.92, ipTM 0.89). Especially, the dimerization interface of the AriB protein is predicted with high confidence values, while local features of the tRNA are not predicted as confidently according to the predicted local difference distance test (pLDDT) (electronic supplementary material, figure S4A). Nonetheless, the overall structure and placement of the tRNA with respect to the protein dimer were identical between many runs, with very small errors in the predicted alignment error (pAE) matrices (electronic supplementary material, figure S4A, right). We were intrigued by this prediction of a symmetric protein dimer interfacing with an asymmetric RNA molecule (figure 4A). Strikingly, in the prediction, one of the AriB monomer nuclease catalytic site is clearly positioned in front of the nucleotides constituting the cleavage site (C40–A41). This increased our confidence in the AF3 model, and we decided to analyse its features in more detail and design experiments to validate the model. Interestingly, two lysine residues (K60 and K64) located on a small alpha-helix seem to hold the anticodon loop above the cleavage site, like tweezers (figure 4B, left, PSE file, electronic supplementary material). Indeed, mutation of these lysines to alanine residues abolished defence against phage T7 by PARIS (figure 4B, right). This experimental observation agrees with the AF3 structure, suggesting a stabilization of the anticodon loop by the lysines K60 and K64 of the AriB monomer that displays cleavage. We then looked at the second catalytic site, wondering why the AriB dimer introduces only a single nick. The second catalytic site faces two nucleotides (U16–U17) of
the D-loop, which are represented by dihydrouridines in the native tRNA. By contrast to C40–A41 in the active catalytic site, AF3 models the U16–U17 nucleotides in a flipped position and interacting with positively charged residues of AriB (R28–U16 and R236–U17), in an arrangement that is apparently not compatible with a cleavage reaction (PSE file, electronic supplementary material). Of note, the nucleotide U16 is present in *E. coli* tRNA<sup>Lys(UUU)</sup> and tRNA<sup>Thr(UGU)</sup> but is substituted by a cytosine in T5 (electronic supplementary material, figure S3D). Introducing the Mut3 (U16→C) substitution in *E. coli* tRNA<sup>Lys(UUU)</sup> significantly impaired AriB cleavage (figure 3D). This suggests that the non-active AriB monomer makes base-specific interactions with the D-loop.

### (f) Cleavage mechanism of AriB

The catalytic site of AriB, as we proposed earlier based on the cryo-electron microscopy (cryoEM) structure of the PARIS complex [13], should be a two-metal-ion ribonuclease. We looked for structural homologues of AriB and found that the AriB catalytic site has structural similarity to the M5 Toprim nuclease, which has a bi-metal cluster with a shared hydroxyl in its active site (figure 4C) [45]. The key amino acid side chains to coordinate the metal cluster are conserved between M5 RNase and AriB, supporting the notion that AriB is a two-metal-ion ribonuclease (figure 4D).

We tested the metal dependency of the nuclease, and found Mg²⁺, Mn²⁺ and Co²⁺ supporting AriB-mediated tRNase activity (electronic supplementary material, figure S4B). Interestingly, the Toprim OLD nuclease has also been reported to be a two-metal-ion enzyme activated by Co²⁺ as well as Mg²⁺, Mn²⁺ and Ca²⁺ for DNA cleavage [24]. In the cleavage reaction of a two-metal-ion nuclease, one metal ion activates water into a nucleophile to attack the nucleic acid phosphate group, breaking the phosphodiester bond, while the other stabilizes the transition state. This reaction is expected to leave a 5'-phosphate end and a 3'-hydroxyl tRNA end after cleavage, as previously depicted [46]. We confirmed the presence of a 3'-OH end by incubating the *in vitro* cleaved tRNA<sup>Lys(UUU)</sup> transcript in the presence of ATP and poly(A) polymerase, which showed the strong shift in the gel expected after polyadenylation of a 3'-OH RNA substrate (figure 4E). We further identified cleavage products using mass spectrometry. AriB reaction products were treated with T1 RNase, which should produce a 14 bp fragment containing the AriB cleavage site in the case of the intact tRNA<sup>Lys(UUU)</sup>, while AriB-mediated nicking between nucleotides 40 and 41 should result in the production of two smaller fragments after T1 cleavage (electronic supplementary material, figure S4C). Indeed, we confirmed the production of the smaller fragments after treatment with AriB WT but not with the AriB E26A mutant. Identified masses of these fragments support the formation of 3'-OH, and 5'-PO₄ products (electronic supplementary material, figure S4D), in line with AriB being a two-metal-ion endoribonuclease.

## 4. Discussion

Toprim nuclease domains have now been found in diverse anti-phage defence systems, where their function remains largely unknown. Here, we investigated the mechanism of the AriB Toprim nuclease from the PARIS bacterial immune system. Upon detection of phage trigger proteins by the AriA ATPase sensor, AriB is released as a dimer to cleave specific host tRNAs, disrupting translation and inhibiting phage replication. Using a combination of tRNA sequencing, *in vitro* cleavage, and ribosome toeprinting assays, we revealed the full set of tRNAs targeted by the AriB nuclease of *E. coli* strain B185. AriB activation leads to the depletion of the previously described tRNA<sup>Lys(UUU)</sup>, but also of tRNA<sup>Thr(UGU)</sup>, tRNA<sup>Asn(GUU)</sup> and tRNA<sup>Thr(CGU)</sup>. All these tRNAs were found to be cleaved between positions 40 and 41, in the stem of the anticodon stem-loop, with tRNA<sup>Lys(UUU)</sup> and tRNA<sup>Thr(UGU)</sup> being the most efficiently targeted tRNAs. We confirmed the biological significance of these tRNA targets by showing that the joint expression of three tRNA variants (tRNA<sup>Lys(UUU)</sup>, tRNA<sup>Thr(UGU)</sup>, tRNA<sup>Asn(GUU)</sup>) from bacteriophage T5 are necessary and sufficient to rescue *E. coli* growth upon PARIS activation. These phage tRNAs all carry nucleotide substitutions at or around the cleavage site of AriB (electronic supplementary material, figure S3C–E), and we showed for *E. coli* tRNA<sup>Lys(UUU)</sup> that mutations at this location indeed abrogate AriB cleavage. Interestingly, the infection of phages blocked by PARIS could sometimes be rescued by the overexpression of just one or two of these tRNAs. This could in some cases be explained by the presence of a variant of the missing tRNA in the genome of the rescued phages, but not always. These experiments reveal differences in the susceptibility of phages to tRNA depletion by PARIS, which remain to be investigated. More experimental data on interactions and cleavage on host tRNAs versus phages tRNAs (electronic supplementary material, figure S3E) could help at understanding the interplay between PARIS defence, phage infection and cellular translation efficiency.

Our results, together with the recent discovery of other anti-phage systems targeting tRNAs, support the hypothesis that phages are under selective pressure to carry diverse tRNAs to overcome bacterial immunity [13,31]. These viral tRNAs may rescue the depletion of the host tRNAs or could also compete with host tRNAs and form non-productive complexes with immune effectors, ensuring that tRNAs are still available for the translation of phage proteins. Other phages may also employ different strategies to counteract tRNA cleavage. For example, the T4 phage employs a kinase (Pnk) and ligase (Rnl1) to repair tRNAs cleaved in the anticodon loop by the PrrC defence system [47].

Using AF3 predictions, we were able to propose a model according to which AriB functions as a symmetric dimer that binds to asymmetric tRNA substrates, resulting in single-site cleavage. This substrate asymmetry forces the two monomers of AriB to adopt distinct interactions with the tRNA. One monomer's catalytic site precisely nicks the anticodon stem, while the second catalytic site can apparently not form a productive substrate complex with the tRNA. Clearly, experimental structures are needed to validate and fully understand this intriguing interaction. Experiments with phage-host chimeric tRNA transcripts suggest that in addition to base-specific contacts in the anticodon stem and the D-loop, AriB likely also makes specific contacts
in other parts of the tRNA, as changing the T5 tRNA<sup>Lys(UUU)</sup> sequence for that of the *E. coli* tRNA<sup>Lys(UUU)</sup> in the anticodon stem loop did not restore cleavage of this tRNA.

A close structural homologue of AriB's catalytic site is the M5 Toprim nuclease, which is involved in ribosomal RNA maturation and cleaves a double-stranded RNA (dsRNA) helix [48]. Unlike AriB, M5 acts as a monomer, cleaving both RNA strands sequentially by repositioning its nuclease domain for the second cleavage [45]. These differences highlight how Toprim domains have evolved different properties, but interestingly the first cleavage site of M5 is identical to that of AriB (5'-CA-3'), leaving 3'- hydroxyl and 5'-phosphate ends after cleavage.

The catalytic activity of other Toprim domain proteins involved in anti-phage defence has also been investigated. This includes the OLD nuclease from bacteriophage P2, which has been studied since the 1970s for its role in blocking infection by phage lambda [49]. Biochemical characterization of the OLD nuclease revealed exonuclease activity on dsDNA as well as nuclease activity on ssDNA and ssRNA [50]. Similar activities, including nicking, were more recently found for other variants of this protein, and their crystal structures determined [24,51-53]. In addition to OLD, the Gabija bacterial immune system, known as class 2 OLD, also carries a Toprim domain in the GajA protein (electronic supplementary material, figure S4E). The Gabija complex was described to cleave dsDNA *in vitro* by action of a GajA dimer nicking each DNA strand at a specific sequence motif [25,54,55]. Further work will be needed to understand the molecular basis of the differences in substrate specificity between AriB, OLD and GajA. We also cannot exclude that some of these proteins might behave differently *in vivo* and *in vitro*, where parameters such as salt and metal concentrations could produce artefacts. An early article on the P2 OLD system suggested that this protein actually targets *E. coli* tRNAs [56]. While this question has not been reinvestigated since the 1970s, confirming these results with modern techniques would help reconcile our findings on AriB with the mechanism of other OLD nucleases.

Finally, several aspects of the AriB mode of action are still not understood. This includes how AriB discriminates between
target and non-target tRNAs. It will be interesting to understand whether the list of targets is conserved across different PARIS
systems (13), and possibly across other Toprim nucleases, or whether AriB is under selective pressure to diversify its substrate
specificity to escape phage countermeasures. Another aspect relates to the activation mechanism of AriB. Our previous data
support the hypothesis that AriB is activated by AriA, and not simply released, after a phage protein is sensed [13]. The
mechanism of this activation remains to be elucidated. Finally, could tRNA-derived small RNA (tsRNA) [57] generated by AriB
be involved in other downstream reactions? We still do not understand the mechanistic consequences of tRNA cleavage by
AriB. While the depletion of the pool of specific tRNAs will naturally lead to translational arrest, we previously showed that
bacteria lose their membrane integrity within 20 min of AriB activation [13]. The chain of events leading to this, and whether or
not bacteria might recover from AriB activation also remain to be investigated.

**Ethics.** This work did not require ethical approval from a human subject or animal welfare committee.

**Data accessibility.** tRNA sequencing data: BioProject ID PRJNA1213714 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1213714).

Electronic supplementary material is available online [58].

**Declaration of AI use.** We have not used AI-assisted technologies in creating this article.

**Authors' contributions.** S.B.: conceptualization, investigation, methodology; B.S.: investigation, methodology, visualization, writing—review and editing; F.D.: investigation, methodology, writing—review and editing; T.L.: investigation; T.P.M.: investigation, methodology; A.L.: investigation, methodology; M.S.: investigation, methodology; M.A.: investigation, methodology; E.U.: investigation, methodology; N.B.: investigation, methodology; P.S.: methodology, supervision, validation; M.L.: investigation; S.D.: investigation, resources; F.B.: funding acquisition, resources, supervision, writing—review and editing; Y.M.: investigation, methodology, validation, visualization, writing—review and editing; V.M.: funding acquisition, investigation, methodology, supervision, validation; G.H.: formal analysis, investigation, validation, visualization, writing—review and editing; A.I.: conceptualization, formal analysis, funding acquisition, methodology, project administration, resources, supervision, validation, visualization, writing—original draft; D.B.: conceptualization, formal analysis, funding acquisition, project administration, resources, supervision, validation, writing—original draft; C.R.: conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, supervision, validation, visualization, writing—original draft.

All authors gave final approval for publication and agreed to be held accountable for the work performed herein.

**Conflict of interest declaration.** We declare we have no competing interests.

**Funding.** A.I. was funded by an RSF grant (24-74-10089). A.L. was supported by a grant from the Russian Science Foundation (24-14-00181). N.B. and P.S. were funded by a Ministry of Science and Higher Education grant (075-15-2024-630). D.B. and C.R. were supported by the European Research Council (no. 101044479) and Agence Nationale de la Recherche (ANR-10-LABX-62-IBEID). S.D., M.L. and F.B. were supported by Agence Nationale de la Recherche (ANR22 CE44 0012 03 SULFO-TRNA).

**Acknowledgements.** We thank Valérie de Crécy-Lagard for discussion on tRNA modifications and Blake Wiedenheft for giving feedback on the manuscript.

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