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:

Genome editing of upstream open reading frames enables translational control in plants

Nature Biotechnology volume 36, pages 894–898 (2018)Cite this article

Subjects

Abstract

Translational regulation by upstream open reading frames (uORFs) is becoming established as a general mechanism for controlling the amount of protein that is synthesized from downstream primary ORFs (pORFs)1,2,3,4,5. We found that genome editing of endogenous uORFs in plants enabled the modulation of translation of mRNAs from four pORFs that are involved in either development or antioxidant biosynthesis. A single-guide RNA that targeted the region harboring a uORF initiation codon can produce multiple mutations. Following uORF editing, we observed varying amounts of mRNA translation in four pORFs. Notably, editing the uORF of LsGGP2, which encodes a key enzyme in vitamin C biosynthesis in lettuce, not only increased oxidation stress tolerance, but also increased ascorbate content by ∼150%. These data indicate that editing plant uORFs provides a generalizable, efficient method for manipulating translation of mRNA that could be applied to dissect biological mechanisms and improve crops.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: CRISPR/Cas9-mediated genome editing of uORFs.
Figure 2: Effect of editing the AtBRI1 uORF.
Figure 3: Editing uORFs of Arabidopsis AtVTC2 and lettuce homologs LsGGP1 and LsGGP2 increases foliar ascorbic acid production.

Similar content being viewed by others

References

  1. Calvo, S.E., Pagliarini, D.J. & Mootha, V.K. Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. Proc. Natl. Acad. Sci. USA 106, 7507–7512 (2009).

    Article  CAS  Google Scholar 

  2. von Arnim, A.G., Jia, Q. & Vaughn, J.N. Regulation of plant translation by upstream open reading frames. Plant Sci. 214, 1–12 (2014).

    Article  CAS  Google Scholar 

  3. McGillivray, P. et al. A comprehensive catalog of predicted functional upstream open reading frames in humans. Nucleic Acids Res. 46, 3326–3338 (2018).

    Article  CAS  Google Scholar 

  4. Liang, X.H. et al. Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames. Nat. Biotechnol. 34, 875–880 (2016).

    Article  CAS  Google Scholar 

  5. Xu, G. et al. uORF-mediated translation allows engineered plant disease resistance without fitness costs. Nature 545, 491–494 (2017).

    Article  CAS  Google Scholar 

  6. Schwanhäusser, B. et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011).

    Article  Google Scholar 

  7. Pelletier, J., Graff, J., Ruggero, D. & Sonenberg, N. Targeting the eIF4F translation initiation complex: a critical nexus for cancer development. Cancer Res. 75, 250–263 (2015).

    Article  CAS  Google Scholar 

  8. Carrieri, C. et al. Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat. Nature 491, 454–457 (2012).

    Article  CAS  Google Scholar 

  9. Zucchelli, S., Patrucco, L., Persichetti, F., Gustincich, S. & Cotella, D. Engineering translation in mammalian cell factories to increase protein yield: the unexpected use of long non-coding SINEUP RNAs. Comput. Struct. Biotechnol. J. 14, 404–410 (2016).

    Article  CAS  Google Scholar 

  10. Simon, A.E. & Miller, W.A. 3′ cap-independent translation enhancers of plant viruses. Annu. Rev. Microbiol. 67, 21–42 (2013).

    Article  CAS  Google Scholar 

  11. Vivinus, S. et al. An element within the 5′ untranslated region of human Hsp70 mRNA which acts as a general enhancer of mRNA translation. Eur. J. Biochem. 268, 1908–1917 (2001).

    Article  CAS  Google Scholar 

  12. Pfeiffer, B.D., Truman, J.W. & Rubin, G.M. Using translational enhancers to increase transgene expression in Drosophila. Proc. Natl. Acad. Sci. USA 109, 6626–6631 (2012).

    Article  CAS  Google Scholar 

  13. Yin, K., Gao, C. & Qiu, J.L. Progress and prospects in plant genome editing. Nat. Plants 3, 17107 (2017).

    Article  CAS  Google Scholar 

  14. Wang, Z.Y., Seto, H., Fujioka, S., Yoshida, S. & Chory, J. BRI1 is a critical component of a plasma-membrane receptor for plant steroids. Nature 410, 380–383 (2001).

    Article  CAS  Google Scholar 

  15. Laing, W.A. et al. An upstream open reading frame is essential for feedback regulation of ascorbate biosynthesis in Arabidopsis. Plant Cell 27, 772–786 (2015).

    Article  CAS  Google Scholar 

  16. Hellens, R.P. et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1, 13 (2005).

    Article  Google Scholar 

  17. Agius, F. et al. Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase. Nat. Biotechnol. 21, 177–181 (2003).

    Article  CAS  Google Scholar 

  18. Bulley, S. et al. Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase. Plant Biotechnol. J. 10, 390–397 (2012).

    Article  CAS  Google Scholar 

  19. Reyes-Chin-Wo, S. et al. Genome assembly with in vitro proximity ligation data and whole-genome triplication in lettuce. Nat. Commun. 8, 14953 (2017).

    Article  CAS  Google Scholar 

  20. Qian, W. et al. Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana. Plant J. 49, 399–413 (2007).

    Article  CAS  Google Scholar 

  21. Shan, Q., Wang, Y., Li, J. & Gao, C. Genome editing in rice and wheat using the CRISPR/Cas system. Nat. Protoc. 9, 2395–2410 (2014).

    Article  CAS  Google Scholar 

  22. Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A. & Liu, D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Article  CAS  Google Scholar 

  23. Gaudelli, N.M. et al. Programmable base editing of A.T to G.C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    Article  CAS  Google Scholar 

  24. Hu, J.H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).

    Article  CAS  Google Scholar 

  25. Yan, L. et al. High-efficiency genome editing in Arabidopsis using YAO promoter-driven CRISPR/Cas9 system. Mol. Plant 8, 1820–1823 (2015).

    Article  CAS  Google Scholar 

  26. Xing, H.L. et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 14, 327 (2014).

    Article  Google Scholar 

  27. Zhai, Z., Jung, H.I. & Vatamaniuk, O.K. Isolation of protoplasts from tissues of 14-d-old seedlings of Arabidopsis thaliana. J. Vis. Exp. 30, e1149 (2009).

    Google Scholar 

  28. Woo, J.W. et al. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat. Biotechnol. 33, 1162–1164 (2015).

    Article  CAS  Google Scholar 

  29. Yoo, S.D., Cho, Y.H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572 (2007).

    Article  CAS  Google Scholar 

  30. Espinosa-Ruiz, A., Martínez, C. & Prat, S. Protocol to treat seedlings with brassinazole and measure hypocotyl length in Arabidopsis thaliana. Bio Protoc. 5, e1568 (2015).

    Article  Google Scholar 

  31. Cui, F. et al. Arabidopsis ubiquitin conjugase UBC32 is an ERAD component that functions in brassinosteroid-mediated salt stress tolerance. Plant Cell 24, 233–244 (2012).

    Article  CAS  Google Scholar 

  32. Zhang, H. et al. The RING finger ubiquitin E3 ligase SDIR1 targets SDIR1-INTERACTING PROTEIN1 for degradation to modulate the salt stress response and ABA signaling in Arabidopsis. Plant Cell 27, 214–227 (2015).

    Article  CAS  Google Scholar 

  33. Kovács, L. et al. Quantitative determination of ascorbate from the green alga Chlamydomonas reinhardtii by HPLC. Bio Protoc. 6, e2067 (2016).

    Article  Google Scholar 

  34. Bae, S., Park, J. & Kim, J.S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J.-L. Qui (Institute of Microbiology) for helpful discussion and insightful comments on the manuscript, Y. Wang and G. Wang for advice on the high-performance liquid chromatography assay, and Q. Xie (Institute of Genetics and Developmental Biology) for providing antibodies to BRI1 and PAG. This work was supported by grants from the National Key Research and Development Program of China (2016YFD0101804), the National Natural Science Foundation of China (31788103, 31420103912, 31501376 and 31570369), and the Chinese Academy of Sciences (QYZDY-SSW-SMC030 and GJHZ1602).

Author information

Author notes

  1. Huawei Zhang, Xiaomin Si and Xiang Ji: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Huawei Zhang, Xiaomin Si, Xiang Ji, Jinxing Liu, Kunling Chen, Daowen Wang & Caixia Gao

  2. University of Chinese Academy of Sciences, Beijing, China

    Xiaomin Si, Xiang Ji, Rong Fan, Daowen Wang & Caixia Gao

  3. Hebei Laboratory of Agricultural Water-Saving, Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang, China

    Rong Fan

Authors
  1. Huawei Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaomin Si
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiang Ji
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rong Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jinxing Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kunling Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Daowen Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Caixia Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.Z., X.S., X.J. and C.G. designed the experiments. X.S., H.Z., X.J., R.F. and J.L. performed the experiments. X.J. and K.C. analyzed the results. C.G. supervised the project and C.G., D.W. and H.Z. wrote the manuscript.

Corresponding author

Correspondence to Caixia Gao.

Ethics declarations

Competing interests

The authors have submitted a patent application (application number 201710976945.0) based on the results reported in this paper.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14 and Supplementary Tables 1–7 (PDF 7506 kb)

Life Sciences Reporting Summary (PDF 130 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, H., Si, X., Ji, X. et al. Genome editing of upstream open reading frames enables translational control in plants. Nat Biotechnol 36, 894–898 (2018). https://doi.org/10.1038/nbt.4202

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.4202

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research