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In Vitro Analysis of ARGONAUTE-Mediated Target Cleavage and Translational Repression in Plants

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Plant Argonaute Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1640))

Abstract

MicroRNAs (miRNAs) are endogenous small RNAs, which negatively regulate expression of complementary target genes at the post-transcriptional level. In plants, miRNAs are mainly loaded onto ARGONAUTE1 to form RNA-induced silencing complexes (RISCs), which mediate target mRNA cleavage as well as translational repression. The cell-free system derived from tobacco BY-2 protoplasts has become a powerful tool not only for the analysis of RISC assembly mechanism but also for mechanistic dissection of plant RISC functions. Here we describe the detailed protocols for the preparation of BY-2 cell lysate and the procedure to analyze the dual function of plant RISC—target cleavage and translational repression—in vitro.

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References

  1. Kozomara A, Griffiths-Jones S (2014) miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 42(Database issue):D68–D73. doi:10.1093/nar/gkt1181

    Article  CAS  PubMed  Google Scholar 

  2. Sunkar R, Li YF, Jagadeeswaran G (2012) Functions of microRNAs in plant stress responses. Trends Plant Sci 17(4):196–203. doi:10.1016/j.tplants.2012.01.010

    Article  CAS  PubMed  Google Scholar 

  3. Kurihara Y, Watanabe Y (2004) Arabidopsis micro-RNA biogenesis through dicer-like 1 protein functions. Proc Natl Acad Sci U S A 101(34):12753–12758. doi:10.1073/pnas.0403115101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Yu B, Yang Z, Li J, Minakhina S, Yang M, Padgett RW, Steward R, Chen X (2005) Methylation as a crucial step in plant microRNA biogenesis. Science 307(5711):932–935. doi:10.1126/science.1107130

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Baumberger N, Baulcombe DC (2005) Arabidopsis ARGONAUTE1 is an RNA slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci U S A 102(33):11928–11933. doi:10.1073/pnas.0505461102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Iki T, Yoshikawa M, Nishikiori M, Jaudal MC, Matsumoto-Yokoyama E, Mitsuhara I, Meshi T, Ishikawa M (2010) In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90. Mol Cell 39(2):282–291. doi:10.1016/j.molcel.2010.05.014

    Article  CAS  PubMed  Google Scholar 

  7. Mi S, Cai T, Hu Y, Chen Y, Hodges E, Ni F, Wu L, Li S, Zhou H, Long C, Chen S, Hannon GJ, Qi Y (2008) Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5′ terminal nucleotide. Cell 133(1):116–127. doi:10.1016/j.cell.2008.02.034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Vaucheret H, Vazquez F, Crete P, Bartel DP (2004) The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev 18(10):1187–1197. doi:10.1101/gad.1201404

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Tang G, Reinhart BJ, Bartel DP, Zamore PD (2003) A biochemical framework for RNA silencing in plants. Genes Dev 17(1):49–63. doi:10.1101/gad.1048103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Iwakawa HO, Tomari Y (2013) Molecular insights into microRNA-mediated translational repression in plants. Mol Cell 52(4):591–601. doi:10.1016/j.molcel.2013.10.033

    Article  CAS  PubMed  Google Scholar 

  11. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53. doi:10.1146/annurev.arplant.57.032905.105218

    Article  CAS  PubMed  Google Scholar 

  12. Todesco M, Rubio-Somoza I, Paz-Ares J, Weigel D (2010) A collection of target mimics for comprehensive analysis of microRNA function in Arabidopsis thaliana. PLoS Genet 6(7):e1001031. doi:10.1371/journal.pgen.1001031

    Article  PubMed  PubMed Central  Google Scholar 

  13. Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, Weigel D (2005) Specific effects of microRNAs on the plant transcriptome. Dev Cell 8(4):517–527. doi:10.1016/j.devcel.2005.01.018

    Article  CAS  PubMed  Google Scholar 

  14. Chen X (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303(5666):2022–2025. doi:10.1126/science.1088060

    Article  CAS  PubMed  Google Scholar 

  15. Aukerman MJ, Sakai H (2003) Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes. Plant Cell 15(11):2730–2741. doi:10.1105/tpc.016238

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gandikota M, Birkenbihl RP, Hohmann S, Cardon GH, Saedler H, Huijser P (2007) The miRNA156/157 recognition element in the 3′ UTR of the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings. Plant J 49(4):683–693. doi:10.1111/j.1365-313X.2006.02983.x

    Article  CAS  PubMed  Google Scholar 

  17. Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P, Yamamoto YY, Sieburth L, Voinnet O (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320(5880):1185–1190. doi:10.1126/science.1159151

    Article  CAS  PubMed  Google Scholar 

  18. Yang L, Wu G, Poethig RS (2012) Mutations in the GW-repeat protein SUO reveal a developmental function for microRNA-mediated translational repression in Arabidopsis. Proc Natl Acad Sci U S A 109(1):315–320. doi:10.1073/pnas.1114673109

    Article  CAS  PubMed  Google Scholar 

  19. Li S, Liu L, Zhuang X, Yu Y, Liu X, Cui X, Ji L, Pan Z, Cao X, Mo B, Zhang F, Raikhel N, Jiang L, Chen X (2013) MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell 153(3):562–574. doi:10.1016/j.cell.2013.04.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Iwakawa HO, Tomari Y (2015) The functions of microRNAs: mRNA decay and translational repression. Trends Cell Biol 25(11):651–665. doi:10.1016/j.tcb.2015.07.011

    Article  CAS  PubMed  Google Scholar 

  21. Liu Q, Paroo Z (2010) Biochemical principles of small RNA pathways. Annu Rev Biochem 79:295–319. doi:10.1146/annurev.biochem.052208.151733

    Article  CAS  PubMed  Google Scholar 

  22. Qi Y, Denli AM, Hannon GJ (2005) Biochemical specialization within Arabidopsis RNA silencing pathways. Mol Cell 19(3):421–428. doi:10.1016/j.molcel.2005.06.014

    Article  CAS  PubMed  Google Scholar 

  23. Fukudome A, Kanaya A, Egami M, Nakazawa Y, Hiraguri A, Moriyama H, Fukuhara T (2011) Specific requirement of DRB4, a dsRNA-binding protein, for the in vitro dsRNA-cleaving activity of Arabidopsis dicer-like 4. RNA 17(4):750–760. doi:10.1261/rna.2455411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Iki T, Yoshikawa M, Meshi T, Ishikawa M (2012) Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants. EMBO J 31(2):267–278. doi:10.1038/emboj.2011.395

    Article  CAS  PubMed  Google Scholar 

  25. Ye R, Wang W, Iki T, Liu C, Wu Y, Ishikawa M, Zhou X, Qi Y (2012) Cytoplasmic assembly and selective nuclear import of Arabidopsis Argonaute4/siRNA complexes. Mol Cell 46(6):859–870. doi:10.1016/j.molcel.2012.04.013

    Article  CAS  PubMed  Google Scholar 

  26. Endo Y, Iwakawa HO, Tomari Y (2013) Arabidopsis ARGONAUTE7 selects miR390 through multiple checkpoints during RISC assembly. EMBO Rep 14(7):652–658. doi:10.1038/embor.2013.73

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Komoda K, Naito S, Ishikawa M (2004) Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts. Proc Natl Acad Sci U S A 101(7):1863–1867. doi:10.1073/pnas.0307131101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Iwakawa HO, Mizumoto H, Nagano H, Imoto Y, Takigawa K, Sarawaneeyaruk S, Kaido M, Mise K, Okuno T (2008) A viral noncoding RNA generated by cis-element-mediated protection against 5′->3′ RNA decay represses both cap-independent and cap-dependent translation. J Virol 82(20):10162–10174. doi:10.1128/JVI.01027-08

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We are grateful to the members of Tomari laboratory for comments on this manuscript. This work was supported in part by Grants-in-Aid for Scientific Research on Innovative Areas (“Nascent-chain Biology”) 26116003 (to H.I.) and (“Non-coding RNA neo-taxonomy”) 26113007 (to Y.T.), Grant-in-Aid for Young Scientists (A) 16H06159 (to H.I.), and Grant-in-Aid for Challenging Exploratory Research 15K14444 (to H.I.).

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Authors and Affiliations

  1. Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan

    Yukihide Tomari & Hiro-oki Iwakawa

  2. Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan

    Yukihide Tomari & Hiro-oki Iwakawa

Authors
  1. Yukihide Tomari
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  2. Hiro-oki Iwakawa
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Correspondence to Hiro-oki Iwakawa .

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Editors and Affiliations

  1. Instituto de Biología Molecular y Celular de Plantas, (Consejo Superior de Investigaciones Científicas–Universidad Politécnica de Valencia), Valencia, Spain

    Alberto Carbonell

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Tomari, Y., Iwakawa, Ho. (2017). In Vitro Analysis of ARGONAUTE-Mediated Target Cleavage and Translational Repression in Plants. In: Carbonell, A. (eds) Plant Argonaute Proteins. Methods in Molecular Biology, vol 1640. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7165-7_4

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  • DOI: https://doi.org/10.1007/978-1-4939-7165-7_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7164-0

  • Online ISBN: 978-1-4939-7165-7

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