TY - JOUR TI - Single molecule analysis reveals reversible and irreversible steps during spliceosome activation AU - Hoskins, Aaron A AU - Rodgers, Margaret L AU - Friedman, Larry J AU - Gelles, Jeff AU - Moore, Melissa J A2 - Staley, Jonathan P VL - 5 PY - 2016 DA - 2016/05/31 SP - e14166 C1 - eLife 2016;5:e14166 DO - 10.7554/eLife.14166 UR - https://doi.org/10.7554/eLife.14166 AB - 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. KW - splicing KW - spliceosome KW - snRNP KW - single-molecule KW - fluorescence KW - RNA JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -