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In Vitro Formation of Plant RNA-Induced Silencing Complexes Using an Extract of Evacuolated Tobacco Protoplasts

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Plant Argonaute Proteins

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

Abstract

Small RNA-mediated gene silencing is involved in a variety of biological processes among many eukaryotic organisms. The silencing effector, generally referred to as RNA-induced silencing complex (RISC), comprises an ARGONAUTE (AGO) protein and a small single-stranded guide RNA in its core. RISCs recognize target genes containing sequences complementary to the guide RNA and repress their expression transcriptionally or posttranscriptionally. In vitro systems that recapitulate RISC assembly are useful not only to decipher the molecular mechanisms underlying the assembly process itself but also to dissect the downstream silencing pathways mediated by RISCs. Here, we describe a method for in vitro plant RISC assembly, which relies on an extract of evacuolated protoplasts derived from Nicotiana tabacum BY-2 suspension-cultured cells. In this extract, synthetic duplexes of small RNAs are incorporated into AGO proteins that are synthesized by in vitro translation, and then duplex unwinding and selective strand elimination result in formation of mature RISCs.

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References

  1. Kawamata T, Tomari Y (2010) Making RISC. Trends Biochem Sci 35(7):368–376. doi:10.1016/j.tibs.2010.03.009

    Article  CAS  PubMed  Google Scholar 

  2. Fang X, Qi Y (2016) RNAi in plants: an Argonaute-centered view. Plant Cell 28(2):272–285. doi:10.1105/tpc.15.00920

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 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 

  4. 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 

  5. 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 

  6. Qi Y, He X, Wang XJ, Kohany O, Jurka J, Hannon GJ (2006) Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443(7114):1008–1012. doi:10.1038/nature05198

    Article  PubMed  Google Scholar 

  7. Takeda A, Iwasaki S, Watanabe T, Utsumi M, Watanabe Y (2008) The mechanism selecting the guide strand from small RNA duplexes is different among argonaute proteins. Plant Cell Physiol 49(4):493–500. doi:10.1093/pcp/pcn043

    Article  CAS  PubMed  Google Scholar 

  8. Iki T (2017) Messages on small RNA duplexes in plants. J Plant Res 130(1):7–16

    Article  CAS  PubMed  Google Scholar 

  9. Poulsen C, Vaucheret H, Brodersen P (2013) Lessons on RNA silencing mechanisms in plants from eukaryotic argonaute structures. Plant Cell 25(1):22–37. doi:10.1105/tpc.112.105643

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14(7):1605–1619

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 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 

  12. 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 

  13. 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 

  14. Smith MR, Willmann MR, Wu G, Berardini TZ, Moller B, Weijers D, Poethig RS (2009) Cyclophilin 40 is required for microRNA activity in Arabidopsis. Proc Natl Acad Sci U S A 106(13):5424–5429. doi:10.1073/pnas.0812729106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 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 

  16. Schuck J, Gursinsky T, Pantaleo V, Burgyan J, Behrens SE (2013) AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system. Nucleic Acids Res 41(9):5090–5103. doi:10.1093/nar/gkt193

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Yoshikawa M, Iki T, Tsutsui Y, Miyashita K, Poethig RS, Habu Y, Ishikawa M (2013) 3′ fragment of miR173-programmed RISC-cleaved RNA is protected from degradation in a complex with RISC and SGS3. Proc Natl Acad Sci U S A 110(10):4117–4122. doi:10.1073/pnas.1217050110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Taichiro Iki

  2. Division of Plant and Microbial Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 2-1-2 Kannondai, Tsukuba, ITsukuba, 305-8602, Ibaraki, Japan

    Masayuki Ishikawa & Manabu Yoshikawa

Authors
  1. Taichiro Iki
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  2. Masayuki Ishikawa
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  3. Manabu Yoshikawa
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Corresponding author

Correspondence to Manabu Yoshikawa .

Editor information

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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Iki, T., Ishikawa, M., Yoshikawa, M. (2017). In Vitro Formation of Plant RNA-Induced Silencing Complexes Using an Extract of Evacuolated Tobacco Protoplasts. 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_3

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  • DOI: https://doi.org/10.1007/978-1-4939-7165-7_3

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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

  • eBook Packages: Springer Protocols

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