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Detection of MicroRNAs Released from Argonautes

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RNA Chaperones

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2106))

Abstract

The Argonaute (AGO) family of proteins plays an essential role in the process of microRNA (miRNA)-mediated gene silencing. More specifically, they are the only known proteins to associate directly with miRNAs within the RNA-induced silencing complex (RISC). Given the importance of miRNA regulation of the transcriptome and its vast implications for human disease, it is essential to understand the molecular underpinnings of miRNA-AGO interactions. Although there are methods available to investigate mature miRNA decay and loading onto AGO2, no feasible method exists to detail the opposite process: release of miRNA from associated AGO proteins. In this chapter, we describe in detail a methodology derived from biochemical approaches, which can be used to quantify the release of any given miRNA from AGOs.

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References

  1. Wu E et al (2017) A continuum of mRNP complexes in embryonic microRNA-mediated silencing. Nucleic Acids Res 45(4):2081–2098

    CAS  PubMed  Google Scholar 

  2. Gehring NH, Wahle E, Fischer U (2017) Deciphering the mRNP Code: RNA-bound determinants of post-transcriptional gene regulation. Trends Biochem Sci 42:369–382

    Article  CAS  Google Scholar 

  3. Bartel DP (2018) Metazoan microRNAs. Cell 173:20–51

    Article  CAS  Google Scholar 

  4. Jonas S, Izaurralde E (2015) Towards a molecular understanding of microRNA-mediated gene silencing. Nat Rev Genet 16:421–433

    Article  CAS  Google Scholar 

  5. Pasquinelli AE (2012) MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship. Nat Rev Genet 13:271

    Article  CAS  Google Scholar 

  6. Ha M, Kim VN (2014) Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15:509–524

    Article  CAS  Google Scholar 

  7. Kwon SC et al (2016) Structure of human DROSHA. Cell 164:81–90

    Article  CAS  Google Scholar 

  8. Nguyen TA, Park J, Dang TL, Choi Y-G, Kim VN (2018) Microprocessor depends on hemin to recognize the apical loop of primary microRNA. Nucleic Acids Res 46(11):5726–5736

    Article  CAS  Google Scholar 

  9. Lee Y et al (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Yi R, Qin Y, Macara IG, Cullen BR (2003) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17:3011–3016

    Article  CAS  Google Scholar 

  12. Bohnsack MT, Czaplinski K, Gorlich D (2004) Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. RNA 10:185–191

    Article  CAS  Google Scholar 

  13. Zeng Y, Cullen BR (2004) Structural requirements for pre-microRNA binding and nuclear export by Exportin 5. Nucleic Acids Res 32:4776–4785

    Article  CAS  Google Scholar 

  14. Hutvágner G et al (2001) A cellular function for the RNA-interference enzyme dicer in the maturation of the let-7 small temporal RNA. Science 293:834–838

    Article  Google Scholar 

  15. Park J-E et al (2011) Dicer recognizes the 5′ end of RNA for efficient and accurate processing. Nature 475:201

    Article  CAS  Google Scholar 

  16. Tian Y et al (2014) A phosphate-binding pocket within the platform-PAZ-connector helix cassette of human dicer. Mol Cell 53:606–616

    Article  CAS  Google Scholar 

  17. Iwasaki S et al (2010) Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes. Mol Cell 39:292–299

    Article  CAS  Google Scholar 

  18. Naruse K, Matsuura-Suzuki E, Watanabe M, Iwasaki S, Tomari Y (2018) In vitro reconstitution of chaperone-mediated human RISC assembly. RNA 24:6–11

    Article  CAS  Google Scholar 

  19. Matranga C, Tomari Y, Shin C, Bartel DP, Zamore PD (2005) Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123:607–620

    Article  CAS  Google Scholar 

  20. Okamura K, Liu N, Lai EC (2009) Distinct mechanisms for microRNA strand selection by Drosophila Argonautes. Mol Cell 36:431–444

    Article  CAS  Google Scholar 

  21. Jo MH et al (2015) Human Argonaute 2 has diverse reaction pathways on target RNAs. Mol Cell 59:117–124

    Article  CAS  Google Scholar 

  22. Yao C, Sasaki HM, Ueda T, Tomari Y, Tadakuma H (2015) Single-molecule analysis of the target cleavage reaction by the Drosophila RNAi enzyme complex. Mol Cell 59:125–132

    Article  CAS  Google Scholar 

  23. Salomon WE, Jolly SM, Moore MJ, Zamore PD, Serebrov V (2015) Single-molecule imaging reveals that Argonaute reshapes the binding properties of its nucleic acid guides. Cell 162:84–95

    Article  CAS  Google Scholar 

  24. Chandradoss SD, Schirle NT, Szczepaniak M, MacRae IJ, Joo CA (2015) Dynamic search process underlies microRNA targeting. Cell 162:96–107

    Article  CAS  Google Scholar 

  25. Fabian MR, Sonenberg N (2012) The mechanics of miRNA-mediated gene silencing: a look under the hood of miRISC. Nat Struct Mol Biol 19:586–593

    Article  CAS  Google Scholar 

  26. Huntzinger E, Izaurralde E (2011) Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet 12:99–110

    Article  CAS  Google Scholar 

  27. Nakanishi K (2016) Anatomy of RISC: how do small RNAs and chaperones activate Argonaute proteins? Wiley Interdiscip Rev RNA 7:637–660

    Article  CAS  Google Scholar 

  28. Janas MM et al (2012) Alternative RISC assembly: binding and repression of microRNA–mRNA duplexes by human Ago proteins. RNA 18:2041–2055

    Article  CAS  Google Scholar 

  29. Yoon J-H et al (2013) Scaffold function of long non-coding RNA HOTAIR in protein ubiquitination. Nat Commun 4:2939

    Article  Google Scholar 

  30. Yoon JH et al (2015) AUF1 promotes let-7b loading on Argonaute 2. Genes Dev 29:1599–1604

    Article  CAS  Google Scholar 

  31. Min KW et al (2017) AUF1 facilitates microRNA-mediated gene silencing. Nucleic Acids Res 45(10):6064–6073

    Article  CAS  Google Scholar 

  32. Zealy RW, Wrenn SP, Davila S, Min K-W, Yoon J-H (2017) microRNA-binding proteins: specificity and function. Wiley Interdiscip Rev RNA 8:e1414

    Article  Google Scholar 

  33. Baccarini A et al (2011) Kinetic analysis reveals the fate of a microRNA following target regulation in mammalian cells. Curr Biol 21:369–376

    Article  CAS  Google Scholar 

  34. Golden RJ et al (2017) An Argonaute phosphorylation cycle promotes microRNA-mediated silencing. Nature 542:197–202

    Article  CAS  Google Scholar 

  35. Shurtleff MJ, Temoche-Diaz MM, Karfilis KV, Ri S, Schekman R (2016) Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction. eLife 5:e19276

    Article  Google Scholar 

  36. Choi YJ, Yoon J-H, Chang JH (2016) Crystal structure of the N-terminal RNA recognition motif of mRNA decay regulator AUF1. Biomed Res Int 2016:9

    Google Scholar 

  37. Kota V et al (2016) SUMO-modification of the La protein facilitates binding to mRNA in vitro and in cells. PLoS One 11:e0156365

    Article  Google Scholar 

  38. Jee D, Lai EC (2014) Alteration of miRNA activity via context-specific modifications of Argonaute proteins. Trends Cell Biol 24:546–553

    Article  CAS  Google Scholar 

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Acknowledgments

Medical University of South Carolina and Hollings Cancer Center to J.H.Y. 2019 Academic Research Support Program in Gangneung-Wonju National University to K.W.M

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA

    Kyung-Won Min, J. Grayson Evans, Erick C. Won & Je-Hyun Yoon

  2. Department of Biology, College of Natural Sciences, Gangneung-Wonju National University, Gangneung, South Korea

    Kyung-Won Min

Authors
  1. Kyung-Won Min
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  2. J. Grayson Evans
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  3. Erick C. Won
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  4. Je-Hyun Yoon
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Corresponding author

Correspondence to Je-Hyun Yoon .

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Editors and Affiliations

  1. Department of Pediatric Hematology, Oncology and Stem Cell Transplantation, University Hospital of Regensburg, Regensburg, Germany

    Tilman Heise

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Min, KW., Evans, J.G., Won, E.C., Yoon, JH. (2020). Detection of MicroRNAs Released from Argonautes. In: Heise, T. (eds) RNA Chaperones. Methods in Molecular Biology, vol 2106. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0231-7_9

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  • DOI: https://doi.org/10.1007/978-1-0716-0231-7_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0230-0

  • Online ISBN: 978-1-0716-0231-7

  • eBook Packages: Springer Protocols

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