Abstract
SummaryThe ATPase Prp16 governs equilibrium between the branching (B∗/C) and exon ligation (C∗/P) conformations of the spliceosome. Here, we present the electron cryomicroscopy reconstruction of the Saccharomyces cerevisiae C-complex spliceosome at 2.8 Å resolution and identify a novel C-complex intermediate (Ci) that elucidates the molecular basis for this equilibrium. The exon-ligation factors Prp18 and Slu7 bind to Ci before ATP hydrolysis by Prp16 can destabilize the branching conformation. Biochemical assays suggest that these pre-bound factors prime the C complex for conversion to C∗ by Prp16. A complete model of the Prp19 complex (NTC) reveals how the branching factors Yju2 and Isy1 are recruited by the NTC before branching. Prp16 remodels Yju2 binding after branching, allowing Yju2 to remain tethered to the NTC in the C∗ complex to promote exon ligation. Our results explain how Prp16 action modulates the dynamic binding of step-specific factors to alternatively stabilize the C or C∗ conformation and establish equilibrium of the catalytic spliceosome.
Highlights
The spliceosome produces mRNA by excising introns from premRNAs in two sequential phosphoryl transfer reactions— branching and exon ligation
We present a detailed analysis of a large electron cryomicroscopy dataset of the C-complex spliceosome from budding yeast
The complete structure of the yeast C-complex spliceosome We previously reported a 3.8-Astructure of the C-complex spliceosome stalled with a 30-splice site (30-SS) mutation and purified by affinity tags on Prp18 and Slu7 (Galej et al, 2016)
Summary
The spliceosome produces mRNA by excising introns from premRNAs in two sequential phosphoryl transfer reactions— branching and exon ligation. The U2 and U6 snRNAs fold into a triple helix conformation that constitutes the active site and allows U6 snRNAs to position two catalytic metals (Fica et al, 2013, 2014; Wilkinson et al, 2020). This active site is stabilized by the binding of the Prp19-associated complex (Galej et al, 2016; Wilkinson et al, 2020) (NTC). The NTC may serve as a hub for the recruitment of some step-specific splicing factors (Chiu et al, 2009; Liu et al, 2007b)
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