Abstract
During pre-mRNA splicing, the spliceosome needs to be activated in an ATP-dependent manner in order to carry out the catalytic steps of the splicing reaction. During activation, the protein composition of the spliceosome changes dramatically and includes loss of the B complex proteins (Spp381, Snu23 and Prp38) and Snu66. Using colocalization single molecule spectroscopy (CoSMoS), we show that Spp381, Snu23, and Snu66 release is dependent on ATP by carrying out reactions at both high and low ATP concentrations. By comparing the characteristic dwell times for these proteins at high ATP, we found that Spp381 and Snu23 behave similar to each other and with kinetics distinct from Snu66. Specifically, at both ATP concentrations, Snu66 shows much longer-lived populations than Spp381 and Snu23. This suggests that Snu66 is bound more stably to the spliceosome than the other two. These results are consistent with previous biochemical and structural studies suggesting that Spp381, Snu23, and Prp38 form a protein subcomplex of the spliceosome. Together, our results suggest that this subcomplex and Snu66 likely have two different roles in the activation step and give rise to multiple activation intermediates as they are sequentially released from the spliceosome.
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