Spliceosomal Pre-mRNA Splicing pp 217-241

Part of the Methods in Molecular Biology book series (MIMB, volume 1126) | Cite as

Single Molecule Approaches for Studying Spliceosome Assembly and Catalysis

  • Eric G. Anderson
  • Aaron A. Hoskins

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.

Key words

Single molecule CoSMoS TIRF Colocalization Fluorescence Microscope Spliceosome Assembly Splicing RNA Ligation SNAP tag 

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Eric G. Anderson
    • 1
  • Aaron A. Hoskins
    • 2
  1. 1.Biochemistry and Molecular PharmacologyUniversity of Massachusetts Medical SchoolWorcesterUSA
  2. 2.Department of BiochemistryUniversity of Wisconsin-MadisonMadisonUSA

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