Investigation of the effects of the splicing inhibitor Pladienolide B and cancer-related mutations in the SF3b1 protein on pre-mRNA splicing in vitro

Pre-mRNA splicing is catalyzed by the spliceosome. A highly dynamic, macromolecular RNP complex is formed out of the U1, U2, U4, U5 and U6 snRNPs and multiple snRNP proteins. During the early stages of spliceosome assembly, the 5’ splice site is first bound by the U1 snRNP. Then during A complex formation, the branch point sequence (BPS) of the intron is recognized and bound by the U2 snRNP. In the subsequent step the U4/U6.U5 tri-snRNP docks generating the pre-B complex and after its stable integration, the pre-catalytic B complex is formed. In the following steps, the spliceosome undergoes compositional and structural rearrangements leading to its catalytic activation which enables the catalysis of the pre-mRNA splicing reaction. SF3b is a heptameric subunit of the U2 snRNP that is required for the stable base pairing of the U2 snRNA with the BPS during A complex assembly, and it plays a key role in the recognition and selection of the branch site adenosine. SF3b1 is a target of many natural pre-mRNA splicing inhibitors, including Pladienolide B (PlaB), which is known to exhibit anti-tumor activity and lead to changes in alternative splicing patterns. In this study I could show that PlaB does not affect the kinetics of cross-intron A complex formation, but prevents the subsequent docking of the tri-snRNP and thus blocking the formation of a pre-B complex. Besides it was observed, that point mutations in the SF3b1 HEAT domain are also linked to various cancer types. A prominent point mutation is the exchange of the lysine at amino acid 700 with glutamic acid (K700E), it is linked to various cancers and leads to the utilization of alternative branch sites and 3' splice sites in vivo. The exact mechanism responsible for these changes in alternative splicing is currently unknown. My studies revealed the nearly complete absence of the U2-associated DEAD-box helicase hPrp5 and a significant reduction in the abundance of the hTAT-SF1 protein, compared to 17S U2 snRNPs containing wild type SF3b1. As hPrp5 plays a key role in the stable binding of U2 during A complex formation, its absence may directly lead to alterations in BPS selection by U2 containing SF3b1K700E. Taken together, my studies provide new insights into the mechanism whereby the SF3b1 modulator PlaB potentially leads to a block in spliceosome assembly, and also for the first time into the effects of SF3b1 cancer-related mutations on the composition of the 17S U2 snRNP. They also pave the way for future in vitro studies to improve our understanding of how alternative branch site usage arises as a consequence of single amino acid substitutions in SF3b1, and thus potentially aid our understanding of how SF3b1 mutations lead to cancer.