Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

RNase III nucleases from diverse kingdoms serve as antiviral effectors

Nature volume 547, pages 114–117 (2017)Cite this article

Subjects

Abstract

In contrast to the DNA-based viruses in prokaryotes, the emergence of eukaryotes provided the necessary compartmentalization and membranous environment for RNA viruses to flourish, creating the need for an RNA-targeting antiviral system1,2. Present day eukaryotes employ at least two main defence strategies that emerged as a result of this viral shift, namely antiviral RNA interference and the interferon system2. Here we demonstrate that Drosha and related RNase III ribonucleases from all three domains of life also elicit a unique RNA-targeting antiviral activity. Systemic evolution of ligands by exponential enrichment of this class of proteins illustrates the recognition of unbranched RNA stem loops. Biochemical analyses reveal that, in this context, Drosha functions as an antiviral clamp, conferring steric hindrance on the RNA-dependent RNA polymerases of diverse positive-stranded RNA viruses. We present evidence for cytoplasmic translocation of RNase III nucleases in response to virus in diverse eukaryotes including plants, arthropods, fish, and mammals. These data implicate RNase III recognition of viral RNA as an antiviral defence that is independent of, and possibly predates, other known eukaryotic antiviral systems.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Drosha mediates miRNA-independent antiviral activity.
Figure 2: The RNA binding domain of Drosha is essential for virus inhibition.
Figure 3: Cytoplasmic Drosha binds stem-loop structures in viral RNA to inhibit RdRp activity.
Figure 4: RNase III inhibition of virus replication is highly conserved.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Koonin, E. V., Wolf, Y. I., Nagasaki, K. & Dolja, V. V. The Big Bang of picorna-like virus evolution antedates the radiation of eukaryotic supergroups. Nat. Rev. Microbiol. 6, 925–939 (2008)

    Article  CAS  Google Scholar 

  2. tenOever, B. R. The evolution of antiviral defense systems. Cell Host Microbe 19, 142–149 (2016)

    Article  CAS  Google Scholar 

  3. tenOever, B. R. RNA viruses and the host microRNA machinery. Nat. Rev. Microbiol. 11, 169–180 (2013)

    Article  CAS  Google Scholar 

  4. Cerutti, H. & Casas-Mollano, J. A. On the origin and functions of RNA-mediated silencing: from protists to man. Curr. Genet. 50, 81–99 (2006)

    Article  CAS  Google Scholar 

  5. Obbard, D. J., Jiggins, F. M., Halligan, D. L. & Little, T. J. Natural selection drives extremely rapid evolution in antiviral RNAi genes. Curr. Biol. 16, 580–585 (2006)

    Article  CAS  Google Scholar 

  6. Shapiro, J. S., Langlois, R. A., Pham, A. M. & Tenoever, B. R. Evidence for a cytoplasmic microprocessor of pri-miRNAs. RNA 18, 1338–1346 (2012)

    Article  CAS  Google Scholar 

  7. Shapiro, J. S., Varble, A., Pham, A. M. & Tenoever, B. R. Noncanonical cytoplasmic processing of viral microRNAs. RNA 16, 2068–2074 (2010)

    Article  CAS  Google Scholar 

  8. Rouha, H., Thurner, C. & Mandl, C. W. Functional microRNA generated from a cytoplasmic RNA virus. Nucleic Acids Res. 38, 8328–8337 (2010)

    Article  CAS  Google Scholar 

  9. Bogerd, H. P., Whisnant, A. W., Kennedy, E. M., Flores, O. & Cullen, B. R. Derivation and characterization of Dicer- and microRNA-deficient human cells. RNA 20, 923–937 (2014)

    Article  CAS  Google Scholar 

  10. Han, J. et al. The Drosha–DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027 (2004)

    Article  CAS  Google Scholar 

  11. Triboulet, R., Chang, H. M., Lapierre, R. J. & Gregory, R. I. Post-transcriptional control of DGCR8 expression by the Microprocessor. RNA 15, 1005–1011 (2009)

    Article  CAS  Google Scholar 

  12. Tang, X., Zhang, Y., Tucker, L. & Ramratnam, B. Phosphorylation of the RNase III enzyme Drosha at serine300 or serine302 is required for its nuclear localization. Nucleic Acids Res. 38, 6610–6619 (2010)

    Article  CAS  Google Scholar 

  13. Court, D. L. et al. RNase III: genetics and function; structure and mechanism. Annu. Rev. Genet. 47, 405–431 (2013)

    Article  CAS  Google Scholar 

  14. Shirako, Y., Strauss, E. G. & Strauss, J. H. Modification of the 5′ terminus of Sindbis virus genomic RNA allows nsP4 RNA polymerases with nonaromatic amino acids at the N terminus to function in RNA replication. J. Virol. 77, 2301–2309 (2003)

    Article  CAS  Google Scholar 

  15. Tsetsarkin, K. A., Liu, G., Shen, K. & Pletnev, A. G. Kissing-loop interaction between 5′ and 3′ ends of tick-borne Langat virus genome ‘bridges the gap’ between mosquito- and tick-borne flaviviruses in mechanisms of viral RNA cyclization: applications for virus attenuation and vaccine development. Nucleic Acids Res. 44, 3330–3350 (2016)

    Article  CAS  Google Scholar 

  16. Bick, M. J. et al. Expression of the zinc-finger antiviral protein inhibits alphavirus replication. J. Virol. 77, 11555–11562 (2003)

    Article  CAS  Google Scholar 

  17. Rupp, J. C., Jundt, N. & Hardy, R. W. Requirement for the amino-terminal domain of sindbis virus nsP4 during virus infection. J. Virol. 85, 3449–3460 (2011)

    Article  CAS  Google Scholar 

  18. Moy, R. H. et al. Stem-loop recognition by DDX17 facilitates miRNA processing and antiviral defense. Cell 158, 764–777 (2014)

    Article  CAS  Google Scholar 

  19. Shapiro, J. S. et al. Drosha as an interferon-independent antiviral factor. Proc. Natl Acad. Sci. USA 111, 7108–7113 (2014)

    Article  CAS  ADS  Google Scholar 

  20. Sabin, L. R. et al. Dicer-2 processes diverse viral RNA species. PLoS ONE 8, e55458 (2013)

    Article  CAS  ADS  Google Scholar 

  21. Sarkar, D., Desalle, R. & Fisher, P. B. Evolution of MDA-5/RIG-I-dependent innate immunity: independent evolution by domain grafting. Proc. Natl Acad. Sci. USA 105, 17040–17045 (2008)

    Article  CAS  ADS  Google Scholar 

  22. Maillard, P. V. et al. Inactivation of the type I interferon pathway reveals long double-stranded RNA-mediated RNA interference in mammalian cells. EMBO J. 35, 2505–2518 (2016)

    Article  CAS  Google Scholar 

  23. Seo, G. J. et al. Reciprocal inhibition between intracellular antiviral signaling and the RNAi machinery in mammalian cells. Cell Host Microbe 14, 435–445 (2013)

    Article  CAS  Google Scholar 

  24. Varble, A. et al. An in vivo RNAi screening approach to identify host determinants of virus replication. Cell Host Microbe 14, 346–356 (2013)

    Article  CAS  Google Scholar 

  25. Cheng, E. H., Levine, B., Boise, L. H., Thompson, C. B. & Hardwick, J. M. Bax-independent inhibition of apoptosis by Bcl-XL. Nature 379, 554–556 (1996)

    Article  CAS  ADS  Google Scholar 

  26. Zhang, F. & Simon, A. E. A novel procedure for the localization of viral RNAs in protoplasts and whole plants. Plant J. 35, 665–673 (2003)

    Article  CAS  Google Scholar 

  27. Perez, J. T. et al. MicroRNA-mediated species-specific attenuation of influenza A virus. Nat. Biotechnol. 27, 572–576 (2009)

    Article  CAS  Google Scholar 

  28. Cherry, S. & Perrimon, N. Entry is a rate-limiting step for viral infection in a Drosophila melanogaster model of pathogenesis. Nat. Immunol. 5, 81–87 (2004)

    Article  CAS  Google Scholar 

  29. Chong, M. M., Rasmussen, J. P., Rudensky, A. Y. & Littman, D. R. The RNAseIII enzyme Drosha is critical in T cells for preventing lethal inflammatory disease. J. Exp. Med. 205, 2005–2017 (2008)

    Article  CAS  Google Scholar 

  30. Ossovskaya, V., Lim, S. T., Ota, N., Schlaepfer, D. D. & Ilic, D. FAK nuclear export signal sequences. FEBS Lett. 582, 2402–2406 (2008)

    Article  CAS  Google Scholar 

  31. Pall, G. S. & Hamilton, A. J. Improved northern blot method for enhanced detection of small RNA. Nat. Protocols 3, 1077–1084 (2008)

    Article  CAS  Google Scholar 

  32. Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011)

    Article  CAS  Google Scholar 

  33. Sedano, C. D. & Sarnow, P. Hepatitis C virus subverts liver-specific miR-122 to protect the viral genome from exoribonuclease Xrn2. Cell Host Microbe 16, 257–264 (2014)

    Article  CAS  Google Scholar 

  34. Manley, J. L. SELEX to identify protein-binding sites on RNA. Cold Spring Harb. Protoc. 2013, 156–163 (2013)

    Article  Google Scholar 

  35. Rio, D. C. Electrophoretic mobility shift assays for RNA-protein complexes. Cold Spring Harb. Protoc. 2014, 435–440 (2014)

    PubMed  Google Scholar 

  36. Lee, Y. et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415–419 (2003)

    Article  CAS  ADS  Google Scholar 

  37. Hardy, R. W. & Rice, C. M. Requirements at the 3′ end of the sindbis virus genome for efficient synthesis of minus-strand RNA. J. Virol. 79, 4630–4639 (2005)

    Article  CAS  Google Scholar 

  38. McCormack, J. C. & Simon, A. E. in Current Protocols in Microbiology Ch. 16, Unit 16D.1 (Wiley, 2006)

  39. Shaffer, S. M., Wu, M. T., Levesque, M. J. & Raj, A. Turbo FISH: a method for rapid single molecule RNA FISH. PLoS ONE 8, e75120 (2013)

    Article  CAS  ADS  Google Scholar 

Download references

Acknowledgements

We thank J. K. Lim and A. G. Pletnev for Langat virus reagents, B. Lee for Sendai virus reagents, R. W. Hardy for SINV reagents, B. R. Cullen for NoDice cells, K. K. Conzelmann for BSR-T7 cells, and B. Ramratnam for pEGFP–Drosha. Recombinant IFN-β was provided by the National Institute of Health’s Biodefense and Emerging Infections Research Resources Repository (HuIFN-β, NR-3080). This material is based upon work supported in part by the Burroughs Wellcome Fund, which provides support for both S.C. and B.R.T. S.C. is also supported by the National Institute of Allergy and Infectious Diseases (NIAID) (R01A1074951). J.P.L. is supported by the DIM Malinf, Conseil Regional d’Ile-de-France. L.C.A. is partly supported by the American Heart Association (15PRE24930012). A.E.S. is supported by National Science Foundation (MCB-1411836) and NIAID (R21AI117882). J.M. is supported by National Institute of General Medicine (F32 GM119235). B.R.T. is also supported by NIAID (R01AI110575).

Author information

Author notes

  1. Lauren C. Aguado and Sonja Schmid: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, 10029, New York, USA

    Lauren C. Aguado, Sonja Schmid, Maryline Panis, Daniel Blanco-Melo & Benjamin R. tenOever

  2. Department of Cell Biology and Molecular Genetics, University of Maryland College Park, College Park, Maryland, 20742, USA

    Jared May & Anne E. Simon

  3. Department of Microbiology, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA

    Leah R. Sabin & Sara Cherry

  4. Department of Pharmacology and Therapeutics, Icahn School of Medicine at Mount Sinai, New York, 10029, New York, USA

    Jaehee V. Shim

  5. Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, 10029, New York, USA

    David Sachs

  6. Macrophages et Développement de l’Immunité, Institut Pasteur, CNRS UMR3738, 25–28 rue du Dr. Roux, Paris, 75724, Cedex 15, France

    Jean-Pierre Levraud

Authors
  1. Lauren C. Aguado
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sonja Schmid
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jared May
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Leah R. Sabin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Maryline Panis
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Daniel Blanco-Melo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jaehee V. Shim
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. David Sachs
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sara Cherry
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Anne E. Simon
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jean-Pierre Levraud
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Benjamin R. tenOever
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.C.A. and S.S. designed and conducted experiments. M.P. performed SELEX. J.M., L.C.A., and A.E.S. were responsible for plant data. L.S. and S.C. generated the Drosophila data. J.V.S. generated the RNaseIII−/− cells. J.P.L. and L.C.A. performed the zebrafish work. D.S. and D.B.M. were responsible for all bioinformatics. B.R.T., L.C.A., and S.S. wrote the paper.

Corresponding author

Correspondence to Benjamin R. tenOever.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks B. Cullen, L. Maraffini and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Characterization of RNaseIII−/− cells.

a, Sequence alignment of genetic alterations in the two alleles encoding Drosha in RNaseIII−/− cells. The deletion and insertion result in a frameshift and early stop codon. b, The ten most abundant miRNAs in each condition, parental HEK293T (WT) or RNaseIII−/− either mock-treated or SINV-infected for 24 h as determined by Illumina small RNA deep sequencing. c, Quantitative PCR (qPCR) analysis of DGCR8 mRNA levels in mock-treated and SINV-infected (MOI = 0.1, 8 h.p.i.) NoDice and RNaseIII−/− cells; error bars, s.d. from three independent experiments; all conditions had P < 0.05 by Student’s t-test. d, Transcriptome profiling and correlation analyses of NoDice cells at baseline and RNaseIII−/− cells transfected with GFP-tagged human Drosha for 72 h. Graph depicts data from two biological replicates per condition.

Extended Data Figure 2 Drosha depletion does not alter the response to IFN-I.

a, qPCR analysis of IFIT1 mRNA levels in NoDice and RNaseIII−/− fibroblasts treated with IFN-β (100 U ml−1, 8 h); error bars, s.d. from three independent experiments; all conditions had P < 0.05 by Student’s t-test. b, Western blot of NoDice and RNaseIII−/− fibroblasts infected with SINV for 1 h before administration of indicated amounts of IFN-β for 24 h. c, d, Northern blot (c) and western blot (d) of primary Rnasenf/f ear-derived fibroblasts treated with indicated AdVs for 2–3 days and then treated with either 100 U IFN-β for 6 h (c) or infected with SINV for 24 h (d). e, mRNA sequencing of total RNA from samples in c. Heatmap depicts known mouse ISGs and IFN downregulated genes with a log2(fold change) greater than 1, as defined by the Interferome database (www.interferome.org).

Extended Data Figure 3 Characterization of Drosha-2A cells.

a, Immunofluorescence of human fibroblasts stably expressing GFP-tagged Drosha-WT or Drosha-2A (S300A/S302A). b, The indicated Flag-tagged proteins were immunoprecipitated from whole-cell extracts (WCE) and incubated at 37 °C with in vitro transcribed genome of SIN124. Production of pre-miR-124 was determined by small RNA northern blot. c, Indicated cell types were infected with SINV at an MOI of 0.001 and viral titres were determined at 16 h.p.i. Shown is the average and s.d. of three independent experiments, with P < 0.05 as determined using a two-tailed Student’s t-test.

Extended Data Figure 4 Drosha-RB-RIIIDmut recognizes stem-loop structures in SINV RNA.

a, Immunoprecipitation of exogenously expressed Flag-tagged proteins. Shown is protein expression in the whole-cell extract and after immunoprecipitation (IP) with a Flag-specific antibody. b, Cells were transfected with Flag-tagged SeV-N or Drosha-RBmut and infected at 36 h.p.t. with SINV at an MOI of 3. At 8 h.p.i., Flag-tagged proteins were immunoprecipitated and bound RNA was isolated to perform qPCR. Graph shows SINV RNA levels relative to input and normalized to tubulin. The average of three independent experiments is shown. Error bars, s.d.; *P < 0.05 using a one-tailed Student’s t-test. c, Prediction of the structure of the 5′ 200 nucleotides of the SINV genome using RNAfold. d, EMSA was performed with the indicated immunoprecipitated proteins and radio-labelled in vitro transcribed RNA comprising the 5′ 200 nucleotides of the SINV genome. Unbound genome is indicated as ‘Free RNA’.

Extended Data Figure 5 Using virus engineering to discern Drosha’s antiviral mechanism.

a, Schematic of the SINV replicon encoding Gaussia luciferase in place of the structural polyprotein used in Fig. 3e–g. b, Schematic of the SINV temperature-sensitive mutant (SIN-RdRpts). Star denotes ts point mutant. c, NoDice and RNaseIII−/− cells were infected with virus depicted in b, at an MOI of 10 and incubated at 40 °C, a temperature at which the mutant viral RdRp is completely inactive. Levels of genomic (g) SINV RNA were determined by qPCR at the indicated times after infection. Data are representative of two independent experiments where each condition was done in triplicate; error bars, s.d. d, Schematic of the SINV encoding firefly luciferase in the nsP3 region and an inactive RdRp (SIN-nsP3Luc) e, Graph depicts levels of in vitro translation of firefly luciferase produced from virus in d, in the presence of membrane fractions from control or Drosha-2A. The data shown are the average of three independent experiments; error bars, s.d.

Extended Data Figure 6 Localization of and miRNA production from cytoplasmic viruses in diverse eukaryotes.

a, Zebrafish embryos were inoculated with SINV for 24 h and then analysed by FISH using a probe complementary to the capsid region of the genome. b, qPCR analysis of zebrafish embryos treated with the indicated morpholinos for 2 days (n = 4); error bars, s.d. c, A. thaliana protoplasts were mock-treated or TCV-infected for 40 h and then analysed by FISH using a Cy3-labelled probe complementary to bases 1210–1259 of the TCV genome. d, Quantification of mature miR-124 production from recombinant TCV was performed using the TaqMan miRNA assay on RNA from Fig. 4b. All samples were normalized to endogenous snoR66. Quantifications of each sample were performed in triplicate; error bars, s.d. from two biological replicates.

Extended Data Figure 7 The impact of diverse RNase III members on virus infection.

a, Schematic depicting core domains of human Drosha, C-terminal region of C. intestinalis Drosha, or full-length RNase III of S. pombe, M. maripaludis, and S. pyogenes. Domains depicted include Proline-rich (P-rich), arginine–serine-rich (RS-rich), conserved central domain (CED), RNaseIII domain (RIIID), and double-stranded RNA binding domain (dsRBD). b, Western blots from BSR-T7 cells, co-transfected with the indicated RNase-III-expression plasmids and SeV rescue plasmids encoding SeV-GFP genome, SeV-N, SeV-P, and SeV-L genes. RNase III expression was determined at 48 h.p.t. and virus replication at 72 h.p.t. c, Western blot of DL1 cells treated with indicated dsRNA for 3 days and subsequently infected with SINV (MOI = 1) for 96 h. d, HEK293T (WT) or RNaseIII−/− cells were infected with SINV for 24 h. Graphs depict the number of SINV reads mapping to indicated positions along the viral genomes from the small RNA deep sequencing performed in Extended Data Fig. 1b.

Supplementary information

Supplementary Information

This file contains the uncropped blots and Supplementary Tables 1-2. (PDF 3967 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Aguado, L., Schmid, S., May, J. et al. RNase III nucleases from diverse kingdoms serve as antiviral effectors. Nature 547, 114–117 (2017). https://doi.org/10.1038/nature22990

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature22990

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology