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Comparative Colocalization Single-Molecule Spectroscopy (CoSMoS) with Multiple RNA Species

  • Reka A. Haraszti
  • Joerg E. Braun
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Part of the Methods in Molecular Biology book series (MIMB, volume 2113)

Abstract

Colocalization single-molecule spectroscopy (CoSMoS) allows studying RNA-protein complexes in the full complexity of their cellular environment at single-molecule resolution. Conventionally, the interaction between a single RNA species and multiple proteins is monitored in real time. However, comparing interactions of the same proteins with different RNA species in the same cell extract promises unique insights into RNA biology. Here, we describe an approach to monitor multiple RNA species simultaneously to enable direct comparison. This approach represents a technological development to avoid conventional inter-experiment comparisons.

Key words

RNA spectroscopy CoSMoS Colocalization single-molecule spectroscopy RNA-protein complex RNP TIRF microscopy Single-molecule spectroscopy Single-molecule fluorescence microscopy 

Notes

Acknowledgments

Joerg E. Braun acknowledges funding from the Human Frontier Science Program (HFSP) LT000166/2013 and the European Molecular Biology Organization (EMBO) ALTF 890-2012. Joerg E. Braun is supported by funding to the laboratory of Melissa J. Moore: NIH R01 GM053007.

References

  1. 1.
    Hoskins AA, Friedman LJ, Gallagher SS, Crawford DJ, Anderson EG, Wombacher R, Ramirez N, Cornish VW, Gelles J, Moore MJ (2011) Ordered and dynamic assembly of single spliceosomes. Science 331(6022):1289–1295.  https://doi.org/10.1126/science.1198830CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Braun JE, Friedman LJ, Gelles J, Moore MJ (2018) Synergistic assembly of human pre-spliceosomes across introns and exons. Elife 7.  https://doi.org/10.7554/eLife.37751
  3. 3.
    Crawford DJ, Hoskins AA, Friedman LJ, Gelles J, Moore MJ (2008) Visualizing the splicing of single pre-mRNA molecules in whole cell extract. RNA 14(1):170–179.  https://doi.org/10.1261/rna.794808CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Loveland AB, Habuchi S, Walter JC, van Oijen AM (2012) A general approach to break the concentration barrier in single-molecule imaging. Nat Methods 9(10):987–992.  https://doi.org/10.1038/nmeth.2174CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Yardimci H, Wang X, Loveland AB, Tappin I, Rudner DZ, Hurwitz J, van Oijen AM, Walter JC (2012) Bypass of a protein barrier by a replicative DNA helicase. Nature 492(7428):205–209.  https://doi.org/10.1038/nature11730CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Crawford DJ, Hoskins AA, Friedman LJ, Gelles J, Moore MJ (2013) Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site. Proc Natl Acad Sci U S A 110(17):6783–6788.  https://doi.org/10.1073/pnas.1219305110CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Lee HW, Kyung T, Yoo J, Kim T, Chung C, Ryu JY, Lee H, Park K, Lee S, Jones WD, Lim DS, Hyeon C, Heo WD, Yoon TY (2013) Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics. Nat Commun 4:1505.  https://doi.org/10.1038/ncomms2507CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lu Y, Wang W, Kirschner MW (2015) Specificity of the anaphase-promoting complex: a single-molecule study. Science 348(6231):1248737.  https://doi.org/10.1126/science.1248737CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    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(1):125–132.  https://doi.org/10.1016/j.molcel.2015.05.015CrossRefPubMedGoogle Scholar
  10. 10.
    Graham TG, Walter JC, Loparo JJ (2016) Two-stage synapsis of DNA ends during non-homologous end joining. Mol Cell 61(6):850–858.  https://doi.org/10.1016/j.molcel.2016.02.010CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Arauz E, Aggarwal V, Jain A, Ha T, Chen J (2016) Single-molecule analysis of lipid-protein interactions in crude cell lysates. Anal Chem 88(8):4269–4276.  https://doi.org/10.1021/acs.analchem.5b04127CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Watanabe M, Iwakawa HO, Tadakuma H, Tomari Y (2017) Biochemical and single-molecule analyses of the RNA silencing suppressing activity of CrPV-1A. Nucleic Acids Res 45(18):10837–10844.  https://doi.org/10.1093/nar/gkx748CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Hoskins AA, Rodgers ML, Friedman LJ, Gelles J, Moore MJ (2016) Single molecule analysis reveals reversible and irreversible steps during spliceosome activation. Elife 5.  https://doi.org/10.7554/eLife.14166
  14. 14.
    Larson JD, Hoskins AA (2017) Dynamics and consequences of spliceosome E complex formation. Elife 6.  https://doi.org/10.7554/eLife.27592
  15. 15.
    Anderson EG, Hoskins AA (2014) Single molecule approaches for studying spliceosome assembly and catalysis. Methods Mol Biol 1126:217–241.  https://doi.org/10.1007/978-1-62703-980-2_17CrossRefPubMedGoogle Scholar
  16. 16.
    Braun JE, Serebrov V (2017) Single-molecule analysis of pre-mRNA splicing with colocalization single-molecule spectroscopy (CoSMoS). Methods Mol Biol 1648:27–37.  https://doi.org/10.1007/978-1-4939-7204-3_3CrossRefPubMedGoogle Scholar
  17. 17.
    Larson JD, Rodgers ML, Hoskins AA (2014) Visualizing cellular machines with colocalization single molecule microscopy. Chem Soc Rev 43(4):1189–1200.  https://doi.org/10.1039/c3cs60208gCrossRefPubMedGoogle Scholar
  18. 18.
    Friedman LJ, Gelles J (2015) Multi-wavelength single-molecule fluorescence analysis of transcription mechanisms. Methods 86:27–36.  https://doi.org/10.1016/j.ymeth.2015.05.026CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Kaur H, Jamalidinan F, Condon SGF, Senes A, Hoskins AA (2019) Analysis of spliceosome dynamics by maximum likelihood fitting of dwell time distributions. Methods 153:13–21.  https://doi.org/10.1016/j.ymeth.2018.11.014CrossRefPubMedGoogle Scholar
  20. 20.
    Larson J, Kirk M, Drier EA, O'Brien W, MacKay JF, Friedman LJ, Hoskins AA (2014) Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope. Nat Protoc 9(10):2317–2328.  https://doi.org/10.1038/nprot.2014.155CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Reka A. Haraszti
    • 1
  • Joerg E. Braun
    • 1
  1. 1.RNA Therapeutics InstituteUniversity of Massachusetts Medical SchoolWorcesterUSA

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