Allosteric small-molecule probes to study splicing switches via conformational control of a spliceosomal RNA helicase

Abstract

Eukaryotic precursor mRNAs contain noncoding regions or introns that must be removed, and the coding sequences or exons are ligated together to generate the mature mRNA. The spliceosome, an elaborate and dynamic multi-megadalton ribonucleoprotein complex, achieves RNA splicing. Alternative splicing enhances the genomes’ coding capacity and allows a quicker response to cellular stimuli via transcriptome-wide adjustments independent of de novo transcription. These transient responses, which are implemented immediately after cellular stimulation via changes in alternative splicing programs, are referred to as immediate early splicing switches. The role of immediate early splicing in controlling switches in gene expression in cell-type specific responses to stimuli has been underexplored. A transient alternative splicing switch upon T-cell activation is implemented via heterogenous ribonucleoprotein C (hnRNPC2 isoform) through transient phosphorylation. This study validated a possible mechanism for the hnRNPC2-phosphorylation-mediated alternative splicing switch in vitro using recombinantly produced hnRNPC WT and its phosphomimetic variant. The primary focus of this study was tool development to gain a deeper understanding of the splicing switches modulated by core spliceosomal components. Multiple highly conserved RNP remodeling enzymes guide and control the compositional and conformational rearrangements of the spliceosome that accompany each splicing event. Among these enzymes, the U5 snRNP-associated human BRR2 (SNRNP200) RNA helicase profoundly remodels the pre-catalytic spliceosome by unwinding U4/U6 di-snRNA to facilitate the transition to the activated spliceosome and therefore was targeted using small-molecule inhibitors. This dual cassette RNA helicase is tightly regulated to ensure splicing fidelity. Cryogenic electron microscopy (cryo-EM), in combination with single-particle analysis of these allosteric inhibitors, bound human BRR2, revealed large, global conformational changes in BRR2, altering the relative position of the helicase cassettes. Our findings underscore the utility of single-particle cryo-EM in uncovering ligand-induced conformational rearrangements that may be obscured in crystal structures and have implications for optimizing compounds that target the dynamic molecular machinery of the spliceosome. The mechanistic understanding of the small-molecule allosteric inhibition supported by high-resolution structures provided the basis for the rational design of next-generation compounds that could be utilized in studying splicing switches.