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Short prokaryotic Argonautes provide defence against incoming mobile genetic elements through NAD+ depletion

Abstract

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). The majority (~60%) of pAgos identified bioinformatically are shorter (comprising only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here we show that Geobacter sulfurreducens short pAgo and the NAD+-bound Sir2 protein form a stable heterodimeric complex. The GsSir2/Ago complex presumably recognizes invading plasmid or phage DNA and activates the Sir2 subunit, which triggers endogenous NAD+ depletion and cell death, and prevents the propagation of invading DNA. We reconstituted NAD+ depletion activity in vitro and showed that activated GsSir2/Ago complex functions as a NADase that hydrolyses NAD+ to ADPR. Thus, short Sir2-associated pAgos provide defence against phages and plasmids, underscoring the diversity of mechanisms of prokaryotic Agos.

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Fig. 1: Sir2/Ago provides defence against phages.
Fig. 2: The GsSir2/Ago system interferes with plasmid transformation.
Fig. 3: The GsSir2 and GsAgo proteins form a heterodimeric complex.
Fig. 4: Nucleic acid binding by GsSir2/Ago in vitro and in vivo.
Fig. 5: GsSir2/Ago binds and hydrolyses NAD+.

Data availability

All data are available in the paper and the supplementary material. In addition, small and total RNA sequencing data are available on the NCBI Sequence Read Archive under BioProject ID PRJNA851009. SAXS data are available in the Small Angle Scattering Biological Data Bank SASBDB under SASBDB ID SASDNH2: https://www.sasbdb.org/data/SASDNH2/. Plasmid sequences used in this work are available at https://www.benchling.com, with exact links for each plasmid provided in Supplementary Table 1.

Geobacter sulfurreducens, Caballeronia cordobensis and Paraburkholderia graminis genomes (respective GenBank accessions: GCA_000210155.1, GCA_001544575.2 and GCA_000172415.1) and all associated sequence and annotation data were obtained from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/). Searches through Pfam (http://pfam.xfam.org/), SwissProt (https://www.expasy.org/resources/uniprotkb-swiss-prot) and PDB (https://www.rcsb.org/) databases were performed. PDB structures mentioned in this study: 5AWH, 4N41, 5UX0, 6LHX, 2H4F.

Code availability

The Julia script used to identify nucleotide frequency at the beginning of the aligned reads and prepare the input for the Weblogo programme is available in the GitHub repository at https://github.com/agrybauskas/argonaute-bound-rna-manuscript.

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Acknowledgements

We thank the Siksnys Laboratory members for their comments on the manuscript and fruitful discussion, and T. de Garay at Refeyn Ltd. for the mass photometry experiments. This work was supported by the European Social Fund (09.3.3-LMT-K-712-01-0126 to V.S.) under a grant agreement with the Research Council of Lithuania (LMTLT), the Israel Science Foundation (grant ISF 296/21 to R.S.), and the Deutsche Forschungsgemeinschaft (SPP 2330, grant 464312965). Funding for open access was provided by Vilnius University.

Author information

Authors and Affiliations

Authors

Contributions

V.S. and M.Z. designed the study; K.T. and C.V. performed bioinformatics and structural modelling; A.L. performed the phage restriction experiments; E.S., D.D., E.G., S.A. and U.T. performed the plasmid transformation experiments; A.S. purified the proteins and performed the SEC-(MALS) experiments; R.G. performed the SEC experiments; E.M. performed the SAXS experiments; E.Z., E.G., D.D. and R.G. performed the EMSA experiments; E.Z. and E.J. reconstituted the GsSir2/Ago-RNA complex and performed the biochemical analysis; A.G. performed the RNA-seq analysis; A.R. performed the mass spectrometry analysis; D.D. performed the NAD+ determination experiments in vivo; E.Z. performed the NAD+ hydrolysis experiments in vitro; R.S., V.S. and M.Z. analysed the data; M.Z. wrote the initial manuscript with input from E.G., R.S., V.S. and other authors. All authors approved the final version.

Corresponding authors

Correspondence to Mindaugas Zaremba or Virginijus Siksnys.

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Competing interests

V.S. is the chairman of CasZyme. R.S. is the scientific founder of BiomX and Ecophage. The remaining authors declare no competing interests.

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Nature Microbiology thanks Malcolm White and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Bioinformatic analysis.

a, PIWI catalytic tetrad DEDX alignment. The 4 catalytic residues (red numbers indicate positions of corresponding GsAgo positions) are shown in 4 motifs of ±3 positions. The motifs are separated by vertical blue lines. Sequence names consist of the following: NCBI sequence ID, abbreviated phylum (for example, ‘ProG’ – gamma-proteobacteria) and organism name. b, Top - a circular phylogenetic tree was generated according to supplementary data provided with Ryazansky et al.8 Long-A pAgo variants are coloured in green (truncated variants without the PAZ domain, light green), long-B pAgo proteins are light green (truncated variants without PAZ, green), and short pAgo proteins are orange. pAgo proteins containing the catalytic tetrad DEDX in their PIWI domain are indicated in blue on the outer circle; pAgos with inactivated PIWI domain are indicated in light grey on the outer circle. pAgo proteins of the GsSir2/Ago, CcSir2/Ago and PgSir2-Ago systems are indicated by ‘Gs’, ‘Cc’ and ‘Pg’, respectively. Bottom - Circular phylogenetic tree of APAZ domains. The circular phylogenetic tree of the five groups of APAZ domains was generated using APAZ domain alignments from Ryazansky et al.8 supplementary file 7. c, Top - Combined alignment of Sir2 domains. Alignment consists of 3 parts, separated by horizontal black lines. In the top part, the Sir2 domain sequences of the GsSir2, CcSir2, PgSir2-Ago and homologues are shown. Logos above depict the conservation of Sir2 domains of Ia and Ib groups. The indicated position numbers correspond to the GsSir2 sequence. In the bottom part, homologues (sirtuins) of catalytically active Thermotoga maritima Sir2 (TmSir2) deacetylase are shown. Logos below indicate the conservation of these homologues. The position numbers correspond to the TmSir2 sequence. Sequences of six motifs that include all positions that form the NAD+-binding pocket, as seen in the TmSir2 structure (PDB ID 2H4F) are shown. Sequence names for the top alignment consist of sequence ID, abbreviated phylum and organism name. Sequence names for bottom alignment all start with ‘Sir2hom’ followed by sequence ID, organism name and short protein name (based on annotation). Stars above the logos indicate residues in the NAD+-binding pocket of canonical sirtuins (for example, TmSir2) that are also conserved. Star colours indicate conservation between the two groups: green – conserved in both canonical sirtuins and GsSir2-like; blue – conserved in both groups, but different; yellow – conserved only in canonical sirtuins; red – conserved only in GsSir2-like proteins. In the middle, alignment of ThsA homologues with Sir2 domains. Bottom - MID domain alignment. Red stars indicate positions of amino acids involved in the binding of the 5’-P end of the guide nucleic acid. The numbering above corresponds to the GsAgo sequence. Additionally, concatenated alignment of just the 6 indicated positions is shown on the right. The three sequences of interest are indicated with red rectangles. Numbers on the left and right of the alignment indicate the first and last positions in the alignment for each sequence.

Extended Data Fig. 2 In vivo characterization of Sir2/Ago systems.

a, Efficiency of plating (EOP) of 4 phages infecting E. coli cells with and without the GsSir2/Ago system from Geobacter sulfurreducens, where the GsSir2/Ago system exhibits no defence activity. The x-axis represents the number of p.f.u. Shown are the means of two replicates in the absence and in the presence of the inducer L-arabinose (L-Ara), with individual data points superimposed. Grey bars represent efficiency of plating (EOP) on pAgo-lacking cells and black bars are EOP in pAgo-containing cells. b, Left - qualitative characterization of plasmid restriction capabilities of PgSir2-Ago system in E. coli strain DH10B. Top: comparison of cell viability in the presence or absence of plasmid-borne PgSir2-Ago expression. Bottom: comparison of plasmid transformation efficiencies in the presence or absence of plasmid-borne PgSir2-Ago expression. Right - qualitative characterization of plasmid restriction capabilities of CcSir2/Ago system in E. coli strain BL21-AI: top - comparison of cell viability in the presence or absence of plasmid-borne CcSir2/Ago expression. Bottom - comparison of plasmid transformation efficiencies in the presence or absence of plasmid-borne CcSir2/Ago expression. c, Quantification of transformation efficiencies for pCDF plasmid with CcSir2/Ago system (three independent replicates, the red line represents average transformation efficiency). d, Cell viability control of BL21-AI, expressing GsSir2/Ago and mutants. e, Control for Fig. 2e – cells contain the pCDF plasmid, however, expression of the GsSir2/Ago system is not induced. f, Qualitative evaluation of pCDF plasmid transformation efficiency in E. coli cells carrying GsSir2/Ago mutants (GsSir2APAZ/Ago, GsSir2/AgoMID and GsSir2/AgoPIWI) of the putative surface of the interaction with nucleic acids. g, Expression analysis of GsSir2/Ago assayed by Western blot. Top: semiquantitative Western blot of the wt GsSir2/Ago complex and its mutants. Numbers above the lanes indicate which part of control protein amount is loaded. The red star shows the lane where the His-tag is on the C-terminus of Ago, rather than the N-terminus of Sir2. GAPDH, loading control. Bottom: Expression analysis of GsSir2/Ago mutants of the putative surface of the interaction with nucleic acids. Three replicates.

Source data

Extended Data Fig. 3 Purification and characterization of Sir2/Ago complexes.

a, MS analysis of wt GsSir2/Ago complex. The theoretical Mw of the GsSir2 and GsAgo proteins (without 1st Met) are 67929.51 Da and 53135.61 Da, respectively. b, CD spectra of wt GsSir2/Ago and mutants. Mutant spectra are similar to that of a natively folded protein. c, Size-exclusion chromatography of wt GsSir2/Ago, showing elution volume and comparing to mass standards. According to mass spectrometry of the purified GsSir2/Ago complex, the molar mass of the GsSir2/Ago heterodimer is 121 kDa. d, SDS–PAGE analysis of fractions containing the CcSir2/Ago complex eluted from Heparin column. Densitometric inspection shows that Sir2 and Ago proteins are in the ratio ~1:1. Single replicate. e, SDS–PAGE analysis of the CcSir2/Ago stock after dialysis against a storage buffer. Various amounts of the stock solution were loaded on the gel. Densitometric inspection shows that Sir2 and Ago proteins are in the ratio ~0.3:1. Single replicate. f, MS analysis of the CcSir2/Ago complex. The experimental masses (53174.71 Da and 67629.21 Da) are close to the theoretical molecular masses of the Ago protein (53173.91 Da) and the Sir2 protein with the truncated tag at the N terminus (67626.24 Da). g, SEC-MALS analysis of the CcSir2/Ago complex. The experimental mass of 124 kDa is close to the theoretical molecular mass of the CcSir2/Ago heterodimer (121 kDa). h, MS/MS calibration curve of NAD+ standard (marked in black) and the observed amount of NAD+ (marked in red) in the CcSir2/Ago complex (20 pmol according to the Ago protein). The discrepancy between the expected amount of NAD+ (20 pmol, marked in green) and the actual amount (6.45 pmol, marked in red) was due to the decrease of the Sir2 protein in the CcSir2/Ago preparation (see e).

Source data

Extended Data Fig. 4 SAXS data.

a, Scattering data on an absolute scale. Linear Guinier plot of the initial part of the scattering curve is in the insert. Points cut from the further processing are shown with empty black symbols. b, Kratky plot, normalized by Rg and I(0) parameters. c, Pair distance distribution function. d, CRYSOL Fit of the scattering curve calculated from the GsSir2/Ago AlphaFold model (black curve) with SAXS data (red points).

Source data

Extended Data Fig. 5 Structural analysis.

a, Comparison of GsSir2/Ago, CcSir2/Ago and PgSir2-Ago AlphaFold models with the X-ray structures of long pAgos. Structures are coloured by domains, schematic domain architecture is given above each structure. Guide RNA and target DNA strands are coloured red and blue, respectively. PDB ID codes for long pAgo structures are given in parentheses. RsAgo represents long-B group, TtAgo – longA group of long pAgos (based on Ryazansky et al. classification8). MpAgo has a distinct OH-type MID domain that is specific to 5’-OH instead of phosphate (MID domain classification – Ryazansky et al.8, MpAgo MID domain biochemical assay - Kaya et al.52). In Sir2/Ago models the N, L1 and L2-like domains previously identified as the APAZ domain correspond to the analogous domains of long pAgos. b, Gs, Cc and Pg Sir2 structural models (cut from full-length models) compared to canonical Sir2 deacetylase TmSir2 and the Sir2 domain of Thoeris defence system protein ThsA. Structures are coloured based on secondary structure. Positions corresponding to ThsA Sir2 N112 and H152 are indicated in green. These residues have been shown to be critical for NAD+ hydrolysis in ThsA24. GsSir2 D230 and corresponding positions in other structures are indicated in cyan. NAD+ was also superimposed on the ThsA Sir2 structure from TmSir2.

Extended Data Fig. 6 EMSA and nucleic acid cleavage experiments.

a, b, Binding of single- and double-stranded oligonucleotides by wt GsSir2/Ago and wt CcSir2/Ago, respectively. A radiolabelled strand indicated by the asterisk. c, Binding of complementary DNA targets by GsSir2/Ago binary complexes pre-loaded with RNA guide containing 5’-phosphate terminus in the presence or absence of heparin. To show that no displacement of the radiolabelled guide by the target strand is observed, a control (Cg*) equivalent to the experimental lane marked by a black triangle, but with the guide, rather than the target, bearing the radioactive label, was performed. d, Control EMSA experiments of ssRNA guide binding by wt GsSir2/Ago (left) and non-complementary DNA target binding by the binary wt GsSir2/Ago-gRNA complex in the presence and absence of heparin. e, Representative binding fit curves of several independent replicates used to calculate Kd of ssRNA guide binding by wt GsSir2/Ago (left) and of target DNA binding by wt GsSir2/Ago-gRNA complex (right). f, (No) cleavage activity of various DNA and RNA oligonucleotides by wt GsSir2/Ago and wt CcSir2/Ago. Reaction products were resolved on a 21% denaturing polyacrylamide gel. In heteroduplexes, the asterisk indicates the radiolabelled strand. For panels A-D and F, at least three independent replicates were performed for each experiment.

Source data

Extended Data Fig. 7 The GsSir2/Ago complex binds NAD+ and causes its depletion.

a, Two ion transitions were used to detect NAD+ in the analysed samples: 662.1→540.1 and 662.1→426.0. b, MS/MS calibration curve of NAD+ standard (marked in black, two replicates) and the observed amount of NAD+ in two samples: 93.7 pmol in the wt GsSir2/Ago sample (marked in green), 0.7 pmol in the sample D230A (marked in red). Black dots represent the means of two replicates and error bars are the standard deviation. c, Detection of NAD+. Comparison of the extracted LC-MS/MS chromatograms: ion transition 662.1→426.0 of wt GsSir2/Ago sample (panel 1) and NAD+ standard (panel 3); ion transition 662.1→540.1 of wt GsSir2/Ago sample (panel 2) and NAD+ standard (panel 4). Green curves - wt GsSir2/Ago sample, grey curves – NAD+ standard. d, Mass chromatogram. For NAD+ detection, an extracted ion current (EIC) for [M-H]- m/z = 662.1018 was used. The comparison of EIC signals shows that the amount of NAD+ in the samples of the non-induced GsSir2/Ago system in the absence (panel 1) and presence (panel 2) of pCDF plasmid is almost the same, while a significant decrease is observed in the sample of the induced GsSir2/Ago system in the absence of pCDF plasmid (panel 3) and only traces of NAD+ are detected in the presence of pCDF plasmid (panel 4).

Source data

Supplementary information

Supplementary Information

Source Data files, Supplementary Note, Methods, References and Tables 1–3.

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Supplementary Data 1

Alignments of sequenced co-purified RNA reads to E. coli genome and vectors.

Source data

Source Data Fig. 1

Numerical data used for graphs in Fig. 1c–f.

Source Data Fig. 2

Numerical data used for graphs.

Source Data Fig. 3

Numerical data used for graphs.

Source Data Fig. 3

Unprocessed gel and blot.

Source Data Fig. 4

Numerical data used for graphs in Fig. 4c,d.

Source Data Fig. 4

Unprocessed images of gels in Fig. 4a,b.

Source Data Fig. 5

Numerical data used for graphs in Fig. 5a–d.

Source Data Extended Data Fig. 2

Numerical data used for graphs in Extended Data Fig. 2a,c.

Source Data Extended Data Fig. 2

Unprocessed images of blots.

Source Data Extended Data Fig. 3

Numerical data used for graphs in Extended Data Fig. 3b,c,g,h.

Source Data Extended Data Fig. 3

Unprocessed images of gels in Extended Data Fig. 3d,e.

Source Data Extended Data Fig. 4

Numerical data used for graphs in Extended Data Fig. 4a–d.

Source Data Extended Data Fig. 6

Numerical data used for graphs.

Source Data Extended Data Fig. 6

Unprocessed images of gels in Extended Data Fig. 6a–d,f.

Source Data Extended Data Fig. 7

Numerical data used for graphs.

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Zaremba, M., Dakineviciene, D., Golovinas, E. et al. Short prokaryotic Argonautes provide defence against incoming mobile genetic elements through NAD+ depletion. Nat Microbiol 7, 1857–1869 (2022). https://doi.org/10.1038/s41564-022-01239-0

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