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Single Molecule Approaches for Studying Spliceosome Assembly and Catalysis

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Spliceosomal Pre-mRNA Splicing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1126))

Abstract

Single molecule assays of splicing and spliceosome assembly can provide unique insights into pre-mRNA processing that complement other technologies. Key to these experiments is the fabrication of fluorescent molecules (pre-mRNAs and spliceosome components) and passivated glass slides for each experiment. Here we describe how to produce fluorescent RNAs by splinted RNA ligation and fluorescent spliceosome subunits by SNAP-tagging proteins in cell lysate. We then depict how to passivate glass slides with polyethylene glycol for use on an inverted microscope with objective-based total internal reflection fluorescence (TIRF) optics. Finally, we describe how to tether the pre-mRNA onto the passivated slide surface and introduce the SNAP-tagged cell lysate for analysis of spliceosome assembly by single molecule fluorescence.

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Acknowledgements

Eric Anderson is supported by funding to the laboratories of Melissa J. Moore (University of Massachusetts Medical School, HHMI Investigator and NIH GM053007) and Jeff Gelles (Brandeis University, NIH GM43369 and GM81648). Aaron Hoskins is funded by startup funds from the University of Wisconsin-Madison and the Wisconsin Alumni Research Foundation and NIH R00 GM079971. We thank Tucker Carrocci, Joshua Larson, and Inna Shcherbakova for critical reading of the manuscript.

Author information

Authors and Affiliations

  1. Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA

    Eric G. Anderson

  2. Department of Biochemistry, University of Wisconsin-Madison, 2214A Biochemical Sciences Building 433 Babcock Dr., Madison, WI, 53706-1544, USA

    Aaron A. Hoskins

Authors
  1. Eric G. Anderson
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  2. Aaron A. Hoskins
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Editor information

Editors and Affiliations

  1. University of California, Irvine, California, USA

    Klemens J. Hertel

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Anderson, E.G., Hoskins, A.A. (2014). Single Molecule Approaches for Studying Spliceosome Assembly and Catalysis. In: Hertel, K. (eds) Spliceosomal Pre-mRNA Splicing. Methods in Molecular Biology, vol 1126. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-980-2_17

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  • DOI: https://doi.org/10.1007/978-1-62703-980-2_17

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-979-6

  • Online ISBN: 978-1-62703-980-2

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