UC Santa Cruz

The spliceosome protein, SF3B1, is associated with U2 snRNP during early spliceosome assembly for pre-mRNA splicing. Frequent somatic mutations in SF3B1 observed in cancer necessitates the characterization of its role in identifying the branchpoint adenosine of introns. Remarkably, SF3B1 is the target of three distinct natural product drugs, each identified by their potent anti-tumor properties. Structural studies indicate that SF3B1 conformational flexibility is functionally important, and suggest that drug binding blocks the transition to a closed state of SF3B1 required for the next stage of spliceosome assembly. This model is confounded, however, by the antagonistic property of an inactive herboxidiene analog. In this study, we established an assay for evaluating the thermostability of SF3B1 present in the nuclear extract preparations employed for in vitro splicing studies, to investigate inhibitor interactions with SF3B1 in a functional context. We show that both active and antagonistic analogs of natural product inhibitors affect SF3B1 thermostability, consistent with binding alone being insufficient to impair SF3B1 function. Surprisingly, SF3B1 thermostability differs among nuclear extract preparations, likely reflecting its conformational status. We also investigated a synthetic SF3B1 ligand, WX-02-23, and found that it increases SF3B1 thermostability and interferes with in vitro splicing by a mechanism that strongly resembles the activity of natural product inhibitors. We propose that altered SF3B1 thermostability can serve as an indicator of inhibitor binding to complement functional assays of their general effect on splicing. It may also provide a means to investigate the factors that influence SF3B1 conformation.