Skip to main content
SpringerLink
Log in

Real-Time Fluorescence Imaging of Single-Molecule Endogenous Noncoding RNA in Living Cells

  • Protocol
RNA Detection

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1649))

Abstract

Visualizing RNA in living cells is increasingly important to facilitate accumulation of knowledge about the relation between specific RNA dynamics and physiological events. Single-molecule fluorescence imaging of target RNAs is an excellent approach to analyzing intracellular RNA motion, but it requires special techniques for probe design and microscope setup. Herein, we present a principle and protocol of an RNA visualization probe based on an RNA binding protein of the Pumilio homology domain (PUM-HD). We also describe the setup and operation of a microscope, and introduce an application to visualize telomeric repeats-containing RNA with telomeres and a telomere-related protein: hnRNPA1. This imaging technique is applicable to visualization of different RNAs, especially including repetitive sequences, in living cells.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 179.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Martin KC, Ephrussi A (2009) mRNA localization: gene expression in the spatial dimension. Cell 136(4):719–730. doi:10.1016/j.cell.2009.01.044. S0092-8674(09)00126-3 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Buxbaum AR, Haimovich G, Singer RH (2015) In the right place at the right time: visualizing and understanding mRNA localization. Nat Rev Mol Cell Biol 16(2):95–109. doi:10.1038/nrm3918. http://www.nature.com/nrm/journal/v16/n2/abs/nrm3918.html#supplementary-information

  3. Geisler S, Coller J (2013) RNA in unexpected places: long non-coding RNA functions in diverse cellular contexts. Nat Rev Mol Cell Biol 14(11):699–712. doi:10.1038/nrm3679

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Heward JA, Lindsay MA (2014) Long non-coding RNAs in the regulation of the immune response. Trends Immunol 35(9):408–419. doi:10.1016/j.it.2014.07.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Engreitz Jesse M, Sirokman K, McDonel P, Shishkin AA, Surka C, Russell P, Grossman Sharon R, Chow Amy Y, Guttman M, Lander Eric S (2014) RNA-RNA interactions enable specific targeting of noncoding RNAs to nascent pre-mRNAs and chromatin sites. Cell 159(1):188–199. doi:10.1016/j.cell.2014.08.018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Oomoto I, Suzuki-Hirano A, Umeshima H, Han Y-W, Yanagisawa H, Carlton P, Harada Y, Kengaku M, Okamoto A, Shimogori T, Wang DO (2015) ECHO-liveFISH: in vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues. Nucleic Acids Res. doi:10.1093/nar/gkv614

  7. Wang X, McLachlan J, Zamore PD, Hall TMT (2002) Modular recognition of RNA by a human pumilio-homology domain. Cell 110(4):501–512. doi:10.1016/S0092-8674(02)00873-5

    Article  CAS  PubMed  Google Scholar 

  8. Cheong CG, Hall TMT (2006) Engineering RNA sequence specificity of pumilio repeats. Proc Natl Acad Sci U S A 103(37):13635–13639. doi:10.1073/pnas.0606294103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yamada T, Yoshimura H, Inaguma A, Ozawa T (2011) Visualization of nonengineered single mRNAs in living cells using genetically encoded fluorescent probes. Anal Chem 83(14):5708–5714. doi:10.1021/ac2009405

    Article  CAS  PubMed  Google Scholar 

  10. Yoshimura H, Inaguma A, Yamada T, Ozawa T (2012) Fluorescent probes for imaging endogenous beta-actin mRNA in living cells using fluorescent protein-tagged pumilio. ACS Chem Biol 7(6):999–1005. doi:10.1021/cb200474a

    Article  CAS  PubMed  Google Scholar 

  11. Yoshimura H, Ozawa T (2016) Chapter 3: Monitoring of RNA dynamics in living cells using PUM-HD and fluorescent protein reconstitution technique. In: Grigory SF, Samie RJ (eds) Methods in enzymology, vol 572. Academic Press, New York, pp 65–85. doi:10.1016/bs.mie.2016.03.018

    Chapter  Google Scholar 

  12. Yamada T, Yoshimura H, Shimada R, Hattori M, Eguchi M, Fujiwara TK, Kusumi A, Ozawa T (2016) Spatiotemporal analysis with a genetically encoded fluorescent RNA probe reveals TERRA function around telomeres. Sci Rep 6:38910. doi:10.1038/srep38910

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Green KM, Linsalata AE, Todd PK (2016) RAN translation—what makes it run? Brain Res 1647:30–42. doi:10.1016/j.brainres.2016.04.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Matsui M, Corey DR (2016) Non-coding RNAs as drug targets. Nat Rev Drug Discov. Advance online publication. doi:10.1038/nrd.2016.117

  15. Faulkner GJ, Kimura Y, Daub CO, Wani S, Plessy C, Irvine KM, Schroder K, Cloonan N, Steptoe AL, Lassmann T, Waki K, Hornig N, Arakawa T, Takahashi H, Kawai J, Forrest ARR, Suzuki H, Hayashizaki Y, Hume DA, Orlando V, Grimmond SM, Carninci P (2009) The regulated retrotransposon transcriptome of mammalian cells. Nat Genet 41(5):563–571. http://www.nature.com/ng/journal/v41/n5/suppinfo/ng.368_s1.html

    Article  CAS  PubMed  Google Scholar 

  16. Vardy L, Orr-Weaver TL (2007) Regulating translation of maternal messages: multiple repression mechanisms. Trends Cell Biol 17(11):547–554. doi:10.1016/j.tcb.2007.09.002

    Article  CAS  PubMed  Google Scholar 

  17. Filipovska A, Razif MF, Nygard KK, Rackham O (2011) A universal code for RNA recognition by PUF proteins. Nat Chem Biol 7(7):425–427. doi:10.1038/nchembio.577

    Article  CAS  PubMed  Google Scholar 

  18. Ozawa T, Yoshimura H, Kim SB (2013) Advances in fluorescence and bioluminescence imaging. Anal Chem 85(2):590–609. doi:10.1021/ac3031724

    Article  CAS  PubMed  Google Scholar 

  19. Yoshimura H, Ozawa T (2014) Methods of split reporter reconstitution for the analysis of biomolecules. Chem Rec 14(3):492–501. doi:10.1002/tcr.201402001

    Article  CAS  PubMed  Google Scholar 

  20. Magliery TJ, Wilson CGM, Pan W, Mishler D, Ghosh I, Hamilton AD, Regan L (2005) Detecting protein−protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism. J Am Chem Soc 127(1):146–157. doi:10.1021/ja046699g

    Article  CAS  PubMed  Google Scholar 

  21. Ozawa T, Nogami S, Sato M, Ohya Y, Umezawa Y (2000) A fluorescent indicator for detecting protein−protein interactions in vivo based on protein splicing. Anal Chem 72(21):5151–5157. doi:10.1021/ac000617z

    Article  CAS  PubMed  Google Scholar 

  22. Tokunaga M, Imamoto N, Sakata-Sogawa K (2008) Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods 5(2):159–161. doi:10.1038/nmeth1171

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, School of Science, The University of Tokyo, Tokyo, 113-0033, Japan

    Hideaki Yoshimura & Takeaki Ozawa

Authors
  1. Hideaki Yoshimura
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Takeaki Ozawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Takeaki Ozawa .

Editor information

Editors and Affiliations

  1. Developmental Biology Unit, EMBL Heidelberg, Heidelberg, Germany

    Imre Gaspar

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Yoshimura, H., Ozawa, T. (2018). Real-Time Fluorescence Imaging of Single-Molecule Endogenous Noncoding RNA in Living Cells. In: Gaspar, I. (eds) RNA Detection. Methods in Molecular Biology, vol 1649. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7213-5_22

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7213-5_22

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7212-8

  • Online ISBN: 978-1-4939-7213-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation