title: Single molecule analysis reveals reversible and irreversible steps during spliceosome activation.
abstract: The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, United States.
authors.forename: Aaron A
authors.identifier: 0000-0002-9777-519X
authors.initials: AA
authors.lastname: Hoskins
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, United States.
authors.forename: Margaret L
authors.initials: ML
authors.lastname: Rodgers
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, United States.
authors.forename: Larry J
authors.identifier: 0000-0003-4946-8731
authors.initials: LJ
authors.lastname: Friedman
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, United States.
authors.forename: Jeff
authors.identifier: 0000-0001-7910-3421
authors.initials: J
authors.lastname: Gelles
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, United States.
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Moore
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D020134:Catalytic Domain; D056004:Molecular Dynamics Simulation; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D000072260:RNA Splicing Factors / Q000235:genetics* / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D000072760:Single Molecule Imaging; D017381:Spliceosomes / Q000235:genetics* / Q000378:metabolism
chemical_list: C079877:PRP19 protein, S cerevisiae; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate
keywords: RNA; S. cerevisiae; biochemistry; biophysics; fluorescence; single-molecule; snRNP; spliceosome; splicing; structural biology