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.

  • Article
  • Published:

Gene regulation by translational inhibition is determined by Dicer partnering proteins

Nature Plants volume 1, Article number: 14027 (2015) Cite this article

Subjects

Abstract

MicroRNAs (miRNAs) are small regulatory RNAs produced by Dicer proteins that regulate gene expression in development and adaptive responses to the environment1–4. In animals, the degree of base pairing between a miRNA and its target messenger RNA seems to determine whether the regulation occurs through cleavage or translation inhibition1. In contrast, the selection of regulatory mechanisms is independent of the degree of mismatch between a plant miRNA and its target transcript5. However, the components and mechanism(s) that determine whether a plant miRNA ultimately regulates its targets by guiding cleavage or translational inhibition are unknown6. Here we show that the form of regulatory action directed by a plant miRNA is determined by DRB2, a DICER-LIKE1 (DCL1) partnering protein. The dependence of DCL1 on DRB1 for miRNA biogenesis is well characterized7–9, but we show that it is only required for miRNA-guided transcript cleavage. We found that DRB2 determines miRNA-guided translational inhibition and represses DRB1 expression, thereby allowing the active selection of miRNA regulatory action. Furthermore, our results reveal that the core silencing proteins ARGONAUTE1 (AGO1) and SERRATE (SE) are highly regulated by miRNA-guided translational inhibition. DRB2 has been remarkably conserved throughout plant evolution, raising the possibility that translational repression is the ancient form of miRNA-directed gene regulation in plants, and that Dicer partnering proteins, such as human TRBP, might play a similar role in other eukaryotic 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: Expression patterns of DRB1 and DRB2 in wild-type plants, and miRNA target expression in drb1 and drb2.
Figure 2: Accumulation of miRNA target mRNAs and proteins in the shoot apex of drb1 and drb2.
Figure 3: Regulation of miRNA targets in floral tissue of drb1 and drb2.
Figure 4: Translational and post-translational regulation of DRB1 and DCL1, and evolutionary conservation of DRB1 and DRB2 proteins.

Similar content being viewed by others

References

  1. Ameres, S. L. & Zamore, P. D. Diversifying microRNA sequence and function. Nature Rev. Mol. Cell Biol. 14, 475–488 (2013).

    Article  CAS  Google Scholar 

  2. Bologna, N. G. & Voinnet, O. Diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu. Rev. Plant Biol. 65, 473–503 (2014).

    Article  CAS  PubMed  Google Scholar 

  3. Sunkar, R., Li, Y-F. & Jagadeeswaran, G. Functions of microRNAs in plant stress responses. Trends Plant Sci. 17, 196–203 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Poethig, R. S. The past, present, and future of vegetative phase change. Plant Physiol. 154, 541–544 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Brodersen, P. et al. Widespread translational inhibition by plant miRNAs and siRNAs. Science 320, 1185–1190 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Mallory, A. & Vaucheret, H. Form, function, and regulation of ARGONAUTE proteins. Plant Cell 22, 3879–3889 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Han, M-H., Goud, S., Song, L. & Fedoroff, N. The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation. Proc. Natl Acad. Sci. USA 101, 1093–1098 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Hiraguri, A. et al. Specific interactions between Dicer-like proteins and HYL1/DRB-family dsRNA-binding proteins in Arabidopsis thaliana. Plant Mol. Biol. 57, 173–188 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Eamens, A. L., Smith, N. A., Curtin, S. J., Wang, M-B. & Waterhouse, P. M. The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes. RNA 15, 2219–2235 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Eamens, A. L., Kim, K. W., Curtin, S. J. & Waterhouse, P. M. DRB2 Is Required for MicroRNA Biogenesis in Arabidopsis thaliana. PLoS ONE 7, e35933 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Curtin, S. J. et al. The roles of plant dsRNA-binding proteins in RNAi-like pathways. FEBS Lett. 582, 2753–2760 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Dugas, D. & Bartel, B. Sucrose induction of Arabidopsis miR398 represses two Cu/Zn superoxide dismutases. Plant Mol. Biol. 67, 403–417 (2008).

    Article  CAS  PubMed  Google Scholar 

  13. Lanet, E. et al. Biochemical evidence for translational repression by Arabidopsis microRNAs. Plant Cell 21, 1762–1768 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Waterhouse, P. M., Wang, M-B. & Lough, T. Gene silencing as an adaptive defence against viruses. Nature 411, 834–842 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. Rajagopalan, R., Vaucheret, H., Trejo, J. & Bartel, D. P. A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev. 20, 3407–3425 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Raja, P., Jackel, J. N., Li, S., Heard, I. M. & Bisaro, D. M. Arabidopsis double-stranded RNA binding protein DRB3 participates in methylation-mediated defense against geminiviruses. J. Virol. JVI, 02305–02313 (2013).

    Google Scholar 

  17. Chen, X. A MicroRNA as a Translational Repressor of APETALA2 in Arabidopsis Flower Development. Science 303, 2022–2025 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Yang, L., Wu, G. & Poethig, R. S. Mutations in the GW-repeat protein SUO reveal a developmental function for microRNA-mediated translational repression in Arabidopsis. Proc. Natl Acad. Sci. USA 109, 315–320 (2012).

    Article  CAS  PubMed  Google Scholar 

  19. Xie, Z., Kasschau, K. D. & Carrington, J. C. Negative feedback regulation of Dicer-like1 in Arabidopsis by microRNA-guided mRNA degradation. Curr. Biol. 13, 784–789 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Eamens, A. L., Wook Kim, K. & Waterhouse, P. M. DRB2, DRB3 and DRB5 function in a non-canonical microRNA pathway in Arabidopsis thaliana. Plant Signal. Behav. 7, 1224–1229 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Floyd, S. K. & Bowman, J. L. Gene regulation: ancient microRNA target sequences in plants. Nature 428, 485–486 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Horman, S. R. et al. Akt-mediated phosphorylation of argonaute 2 downregulates cleavage and upregulates translational repression of MicroRNA targets. Mol. Cell 50, 356–367 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bouquin, T., Mattsson, O., Næsted, H., Foster, R. & Mundy, J. The Arabidopsis lue1 mutant defines a katanin p60 ortholog involved in hormonal control of microtubule orientation during cell growth. J. Cell Sci. 116, 791–801 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Martin, T., Oswald, O. & Graham, I. A. Arabidopsis seedling growth, storage lipid mobilization, and photosynthetic gene expression are regulated by carbon:nitrogen availability. Plant Physiol. 128, 472–481 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Coutu, C., Brandle, J., Brown, D. & Brown, K. pORE: a modular binary vector series suited for both monocot and dicot plant transformation. Transgenic Res. 16, 771–781 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Jefferson, R. A., Kavanagh, T. A. & Bevan, M. W. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901–3907 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Stocks, M. B. et al. The UEA sRNA workbench: a suite of tools for analysing and visualizing next generation sequencing microRNA and small RNA datasets. Bioinformatics 28, 2059–2061 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Skirycz, A. et al. A Reciprocal 15N-labeling proteomic analysis of expanding Arabidopsis leaves subjected to osmotic stress indicates importance of mitochondria in preserving plastid functions. J. Proteome Res. 10, 1018–1029 (2010).

    Article  Google Scholar 

  29. Arsova, B., Zauber, H. & Schulze, W. X. Precision, proteome coverage, and dynamic range of arabidopsis proteome profiling using 15N metabolic labeling and label-free approaches. Mol. Cell. Proteomics 11, 619–628 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Schaff, J. E., Mbeunkui, F., Blackburn, K., Bird, D. M. & Goshe, M. B. SILIP: a novel stable isotope labeling method for in planta quantitative proteomic analysis. Plant J. 56, 840–854 (2008).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank K. Nakasugi for deep sequencing data analysis and D. Barton for GUS imaging, A. Tay for statistical analysis of proteomics data, A.V.R. Ribeiro for graphical assistance and T. Roberts and R. Hellens for critical reading of our manuscript. We acknowledge M. Raftery, L. Zhong and S. Liu Lau for their maintenance of the orbitrap mass spectrometers at the UNSW Bioanalytical Mass Spectrometry Facility. PMW acknowledges support as a Federation Fellow from the Australian Research Council and contributions from the University of Sydney, CSIRO and QUT. M.R.W. acknowledges support from the Australian Research Council, the Australian Federal Government EIF Super Science Scheme and the University of New South Wales. G.H-S. was the recipient of an Australian Research Council Australian Postdoctoral Research Fellowship and funds from the University of New South Wales ECR Scheme. R.S.R. was the recipient of an Australian Postgraduate Award.

Author information

Authors and Affiliations

  1. School of Biological Sciences, University of Sydney, Macleay Building A12, Sydney, 2006, New South Wales, Australia

    Rodrigo S. Reis & Peter M. Waterhouse

  2. Faculty of Agriculture and Environment, University of Sydney, Eveleigh, New South Wales, Australia

    Rodrigo S. Reis

  3. Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, 2052, New South Wales, Australia

    Gene Hart-Smith & Marc R. Wilkins

  4. School of Environmental and Life Sciences, University of Newcastle, Callaghan, 2308, New South Wales, Australia

    Andrew L. Eamens

  5. Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, 4001, Queensland, Australia

    Peter M. Waterhouse

Authors
  1. Rodrigo S. Reis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gene Hart-Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andrew L. Eamens
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marc R. Wilkins
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter M. Waterhouse
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.H-S., R.S.R. and M.R.W. conceived and designed the proteomics experiments; G.H-S. performed the mass spectrometry experiments and data analysis; R.S.R., A.L.E. and P.M.W. conceived all other experiments; A.L.E. performed miR863 northern blots; R.S.R. designed and performed all other experiments; R.S.R. and P.M.W. interpreted the data; R.S.R., A.L.E. and P.M.W. wrote the manuscript.

Corresponding authors

Correspondence to Rodrigo S. Reis or Peter M. Waterhouse.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Reis, R., Hart-Smith, G., Eamens, A. et al. Gene regulation by translational inhibition is determined by Dicer partnering proteins. Nature Plants 1, 14027 (2015). https://doi.org/10.1038/nplants.2014.27

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nplants.2014.27

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

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