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Free keywords:
Cwc24; dcFCCS; Prp2-mediated; spliceosome dynamics; U2 SF3a/b
Abstract:
The compositional and conformational changes during catalytic activation of the spliceosome promoted by the DEAH box
ATPase Prp2 are only poorly understood. Here, we show by dual-color fluorescence cross-correlation spectroscopy (dcFCCS) that
the binding affinity of several proteins is significantly changed during the Prp2-mediated transition of precatalytic Bact
spliceosomes to catalytically activated B* spliceosomes from Saccharomyces cerevisiae. During this step, several proteins,
including the zinc-finger protein Cwc24, are quantitatively displaced from the B* complex. Consistent with this, we show that
Cwc24 is required for step 1 but not for catalysis per se. The U2-associated SF3a and SF3b proteins Prp11 and Cus1 remain bound
to the B* spliceosome under near-physiological conditions, but their binding is reduced at high salt. Conversely, high-affinity
binding sites are created for Yju2 and Cwc25 during catalytic activation, consistent with their requirement for step 1 catalysis.
Our results suggest high cooperativity of multiple Prp2-mediated structural rearrangements at the spliceosome’s catalytic core.
Moreover, dcFCCS represents a powerful tool ideally suited to study quantitatively spliceosomal protein dynamics in equilibrium.
