Identification of herboxidiene structural features that mediate conformation-dependent SF3B1 interactions to inhibit splicing.

Abstract

Small molecules that target the spliceosome SF3B complex are potent inhibitors of cancer cell growth. The compounds affect an early stage of spliceosome assembly when U2 snRNP first engages the branch point sequence of an intron. Recent cryo-EM models of U2 snRNP before and after intron recognition suggest several large-scale rearrangements of RNA and protein interactions involving SF3B. Employing an inactive herboxidiene analog as a competitor with SF3B inhibitors, we present evidence for multiple conformations of SF3B in the U2 snRNP, only some of which are available for productive inhibitor interactions. We propose that both thermodynamics and an ATP-binding event promote the conformation conducive to SF3B inhibitor interactions. However, SF3B inhibitors do not impact an ATP-dependent rearrangement in U2 snRNP that exposes the branch binding sequence for base pairing. We also report extended structure activity relationship analysis of herboxidiene, which identified features of the tetrahydropyran ring that mediate its interactions with SF3B and its ability to interfere with splicing. In combination with structural models of SF3B interactions with inhibitors, our data leads us to extend the model for early spliceosome assembly and inhibitor mechanism. We postulate that interactions between a carboxylic acid substituent of herboxidiene and positively charged SF3B1 sidechains in the inhibitor binding channel are required to maintain inhibitor occupancy and counteract the SF3B transition to a closed state that is promoted by stable U2 snRNA interactions with the intron.