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.
Highlights
The spliceosome is one of the most dynamic molecular machines inside the cell
We have shown that snRNPs and the NTC can dynamically engage pre-mRNAs under ATP concentrations that permit splicing (2 mM) (Hoskins et al, 2011; Shcherbakova et al, 2013)
We wondered if a similar approach could be used to stall spliceosomes to observe B complex on single pre-mRNA molecules and gain insights into the mechanisms of spliceosomal activation in single molecule experiments
Summary
Removal of introns from precursors to mRNAs (pre-mRNAs) involves the coordinated action of 5 small nuclear RNAs (snRNAs) and >100 proteins (Wahl et al, 2009; Hoskins and Moore 2012). Some of these proteins along with the snRNAs assemble into small nuclear ribonucleoprotein particles (the U1, U2, U4, U5, and U6 snRNPs) that work together with other accessory proteins to catalyze splicing. A number of biochemical and genetic experiments have elucidated the splicing factors present at each stage (Wahl et al, 2009; Fabrizio et al, 2009), as well as characteristic interactions between snRNA, pre-mRNA, and protein components (Brow 2002; Wahl et al, 2009)
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