Abstract
RNA interference is widely distributed in eukaryotes and has a variety of functions, including antiviral defence and gene regulation1,2. All RNA interference pathways use small single-stranded RNA (ssRNA) molecules that guide proteins of the Argonaute (Ago) family to complementary ssRNA targets: RNA-guided RNA interference1,2. The role of prokaryotic Ago variants has remained elusive, although bioinformatics analysis has suggested their involvement in host defence3. Here we demonstrate that Ago of the bacterium Thermus thermophilus (TtAgo) acts as a barrier for the uptake and propagation of foreign DNA. In vivo, TtAgo is loaded with 5′-phosphorylated DNA guides, 13–25 nucleotides in length, that are mostly plasmid derived and have a strong bias for a 5′-end deoxycytidine. These small interfering DNAs guide TtAgo to cleave complementary DNA strands. Hence, despite structural homology to its eukaryotic counterparts, TtAgo functions in host defence by DNA-guided DNA interference.
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Accession codes
Accessions
Gene Expression Omnibus
Data deposits
The RNA-seq data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus under accession number GSE52738. The siDNA sequence data discussed in this publication have been deposited in NCBI s BioSample database and are accessible under accession number SAMN02593821.
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Acknowledgements
We want to thank A. Hidalgo, C. E. César, M. Davids and R. H. J. Staals for advice on experimental procedures. Furthermore, we would like to thank R. Engelhart, B. van Genugten, G. Göertz and R. Stolk for experimental contributions. This work was financially supported by grants from the Netherlands Organization of Scientific Research (NWO) to J.O. (NWO-TOP, 854.10.003), and to S.J.J.B. (NWO Vidi , 864.11.005), and by project BIO2010-18875 from the Spanish Ministry of Science and Innovation, and an Institutional Grant from the Fundación Ramón Areces to CBMSO (J.B.).
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Contributions
M.M.J. and J.H.J. made genomic T. thermophilus mutants under the supervision of J.v.d.O. T. thermophilus experiments were performed by D.C.S., M.M.J. and J.H.J. under the supervision of J.B., S.J.J.B. and J.v.d.O. D.C.S. and E.R.W. purified RNA for RNA-seq, and D.C.S. analysed RNA-seq data under the supervision of S.J.J.B. and J.v.d.O. D.C.S. and A.P.S. performed experiments in which TtAgo expression in T. thermophilus was shown using mass spectrometry. D.C.S., M.M.J. and J.H.J. made all plasmid constructs under the supervision of S.J.J.B., J.B. and J.v.d.O. D.C.S., E.R.W. and Y.Z. purified and analysed TtAgo guides. In vitro activity assays were designed and analysed by D.C.S., S.J.J.B., Y.W., D.J.P. and J.v.d.O., and performed by D.C.S. and Y.Z. under the supervision of S.J.J.B. and J.v.d.O. All authors read and approved the submitted manuscript.
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Extended data figures and tables
Extended Data Figure 1 Analyses of TtAgo in T. thermophilus and E.coli.
a, TtAgo decreases plasmid transformation efficiency of T. thermophilus. Transformation efficiency of different ago mutant strains relative to the transformation efficiency of wild-type strain HB27. HB27EC is an HB27 mutant selected for high competence, and HB27Δago is an ago gene knockout strain (Fig. 1a). Strains were transformed with plasmid pMK184 (Extended Data Table 5). Transformations were performed in biological duplicates for each strain. Error bars indicate standard deviations. b, Effect on TtAgo expression on plasmid content in E. coli KRX. TtAgo and TtAgoDM were expressed in E. coli KRX from plasmid pWUR702 and pWUR703. Plasmids were purified from biological triplicate cultures in which expression was induced (+) or not induced (−). Compared with TtAgoDM expression, TtAgo expression in E. coli KRX does not lead to reduced plasmid content. Changes in plasmid yield between induced and not induced cultures probably originate from protein expression energy costs. Error bars indicate standard deviations. c, 10–150-nucleotide (nt) RNA with 5′-OH group co-purifies with TtAgo. 15% denaturing polyacrylamide gels with nucleic acids co-purified with TtAgo and TtAgoDM. Nucleic acids are phosphorylated in a T4 PNK forward reaction (5′-OH groups, and to a lesser extend 5′-P groups, are labelled) using [γ-32P] ATP, and resolved on 15% denaturing polyacrylamide gels. Nucleic acids were not treated (lane 1, 5), RNaseA treated (lanes 2, 6), DNaseI treated (lane 3, 7) or Nuclease P1 treated (lane 4, 8).
Extended Data Figure 2 Change in transcription of T. thermophilus genes after ago gene knockout.
a, b, RNA-seq analysis was performed on biological triplicates for each strain. Change in gene expression of genes encoded on the chromosome (a) or on the megaplasmid (b) is shown as the log2 of the fold difference in expression of the average of normalized mapped reads on that gene in HB27Δago compared with the average of normalized mapped reads on that gene in HB27. Peaks corresponding to genes involved in host defence are coloured red, whereas peaks corresponding to genes involved in competence are coloured blue. c, Genes or operons containing genes with a log2 expression change greater than 2 or −2.
Extended Data Figure 3 TtAgo cleaves ssDNA using ssDNA guides.
a, 21-nucleotide (nt) DNA and RNA guides are complementary to the 45-nucleotide DNA targets. Predicted cleavage positions are indicated with a black triangle. b, 20% denaturing polyacrylamide gel loaded with samples in which TtAgo and TtAgoDM were provided with an RNA or an DNA guide to cleave a 45-nucleotide ssDNA target. c, 21-nucleotide RV and FW DNA guides are complementary to the 98-nucleotide ssDNA targets. Predicted cleavage positions are indicated with a black triangle. d, 98-nucleotide ssDNA targets are incubated with TtAgo and TtAgoDM, provided with complementary and non-complementary DNA guides, and resolved on 15% denaturing polyacrylamide gels.
Extended Data Figure 4 Effect of variation of the 5′-end deoxynucleoside of the siDNA and effect of the temperature on TtAgo cleavage efficiency.
a–d, Cleavage of 98-nucleotide ssDNA target (Extended Data Fig. 3c) by TtAgo loaded with complementary siDNAs containing a different 5′ deoxynucleoside, as shown in red. The concentrations of each siDNA were varied (indicated on top of the gels). Products of the reaction were resolved on 15% denaturing polyacrylamide gels. e, TtAgo expression plasmid pWUR702 (no guides added) incubated with TtAgo and TtAgoDM at different temperatures. f, pWUR708 plasmid (FW and RV guides added; Fig. 4b) incubated with TtAgo and TtAgoDM at different temperatures, resolved on 0.8% agarose gels. LIN, linear; M1, 1 kb Generuler marker (Fermentas); OC, open circular; SC, supercoiled. g, 98-nucleotide RV target cleavage (FW guide added) incubated with TtAgo and TtAgoDM at different temperatures, resolved on a 15% denaturing acrylamide gel. M2, O’RangeRuler 5 bp DNA Ladder (Thermo Scientific).
Extended Data Figure 5 Activity analyses of TtAgo.
a, b, AT-rich (17% GC) insert of pWUR704 (a) and GC-rich insert (59% GC) of pWUR705 (b). The target sequence is boxed. Restriction sites HindIII and BsmI are indicated in grey. Sequences are displayed in the 3′–5′ direction to allow comparison with Fig. 4b, which shows guide base pairing to this sequence. c, d, SpeI-linearized plasmid pWUR704 (c) and pWUR705 (d) incubated with TtAgo–siDNA and TtAgoDM–siDNA complexes targeting both strands of the plasmid, and resolved on 0.8% agarose gels. LIN, linear; M1, 1 kb Generuler marker (Fermentas); M2, open circular and linearized pWUR704 (c), or open circular and linearized pWUR705 (d); OC, open circular. FW guide: BG3466. RV guide: BG4017. High salt concentration (250 mM NaCl) in the reaction buffer cause the TtAgo-treated samples to run higher in the gel than M1 and M2. e, Two-step plasmid cleavage. Target pWUR704 was first nicked by a TtAgo–siDNA complex targeting the first strand (FW guide, lane 1), after which a TtAgo–siDNA complex targeting the other strand was added (RV guide, lane 2). FW guide is still present, and its presence is therefore indicated as (+). LIN, linear; M1, 1 kb Generuler marker (Fermentas); OC, open circular; SC, supercoiled. f, g, Nb.BsmI-nicked plasmid pWUR704 (f) and pWUR705 (g) incubated with TtAgo–siDNA and TtAgoDM–siDNA complexes targeting the un-nicked strands of the plasmid (33 bp away from the nicking site), and resolved on 0.8% agarose gels. LIN, linear; M1, 1 kb Generuler marker (Fermentas); M2, open circular and linearized pWUR704 (a), or open circular and linearized pWUR705 (b); OC, open circular. High salt concentrations (250 mM NaCl) in the reaction buffer cause the TtAgo-treated samples to run higher in the gel than M1 and M2. h, TtAgo dsDNA cleavage site analysis. (i) Plasmid pWUR704 with TtAgo–siDNA target sequences. Predicted cleavage sites are indicated with black triangles. (ii) pWUR704 was linearized using TtAgo–siDNA complexes targeting the plasmid on both strands. (iii) The linearized plasmid was cleaved using either NheI (as shown) or XbaI (not shown). (iv) Restriction site overhangs and possible overhangs resulting from TtAgo–siDNA cleavage were filled using Klenow fragment polymerase (Fermentas). (v) Blunt-end DNA was ligated using T4 DNA ligase (Fermentas), after which the plasmid could be transformed and later sequenced to determine the cleavage site. Sequences revealed that TtAgo–siDNA complexes indeed cleaved the target at the predicted locations (as shown in a), and are shown in more detail in Fig. 4b and Extended Data Fig. 5a, b. Note that in this picture target sequences are displayed in reversed order compared with Fig. 4b and Extended Data Fig. 5a, b. j, TtAgo prefers Mn2+ over Mg2+ as a divalent cation for cleavage. (i) 21-nucleotide DNA guide and 98-nucleotide ssDNA target used. The predicted cleavage site is indicated with a black triangle. (ii) 98-nucleotide ssDNA target cleavage reaction with TtAgo loaded with a 21-nucleotide siDNA in the presence of increasing concentrations of Mg2+, as indicated on top of the gel. (iii) 98-nucleotide ssDNA target cleavage reaction with TtAgo loaded with a 21-nucleotide siDNA in the presence of increasing concentrations of Mn2+, as indicated. Samples were resolved on 15% denaturing polyacrylamide gels.
Supplementary information
Supplementary Data 1
This file contains the Source Data for Extended Data Table 1 and Extended Data Figure 2. (XLSX 572 kb)
Supplementary Data 2
This file contains the Source Data for Extended Data Table 3. (XLSX 8 kb)
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Swarts, D., Jore, M., Westra, E. et al. DNA-guided DNA interference by a prokaryotic Argonaute. Nature 507, 258–261 (2014). https://doi.org/10.1038/nature12971
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DOI: https://doi.org/10.1038/nature12971
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