title: Single molecule approaches for studying spliceosome assembly and catalysis.
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.
authors.affiliation: Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: Eric G
authors.initials: EG
authors.lastname: Anderson
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Hoskins
mesh_terms: D002384:Catalysis; D002457:Cell Extracts; D005453:Fluorescence; D008967:Molecular Biology / Q000379:methods*; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D012441:Saccharomyces cerevisiae; D017381:Spliceosomes / Q000235:genetics* / Q000648:ultrastructure
chemical_list: D002457:Cell Extracts; D012322:RNA Precursors