Structural investigation of splicing regulation under pathological and drug-induced conditions

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Pre-mRNA splicing occurs on the spliceosome, a large protein–RNA complex that removes non-coding introns and ligates coding exons into a mature RNA. The selection and definition of exons for inclusion or omission in spliced RNA is one of the most complex phenomena in molecular biology, and its misregulation constitutes a major cause of pathological disorders in humans.The spliceosomal ribonucleoprotein U2 snRNP stands out as a critical player in splicing regulation, owing to its distinctive positioning on the branch-site region of the pre mRNA. Mutations of the SF3b complex of the U2 snRNP have high incidence rates in various forms of cancer and lead to aberrant splicing via a mechanism that is currently unclear.Moreover, several splicing modulators with antitumor properties, such as pladienolide B, herboxidiene, and spliceostatin A, bind the SF3b complex and inhibit normal branch-site usage, inducing intron retention and exon skipping. Despite a decade of research, the binding sites of modulators and their mechanism of action remain unclear.Our goal is to achieve a breakthrough in understanding the mechanism that enables carcinogenic mutations and antitumor compounds with therapeutic potential to modulate the regulation of splicing. Specifically, we aim to engineer spliceosomal complexes and SF3b variants that capture splicing regulation under pathological and drug-induced conditions, and to characterize their 3D structures by electron cryo-microscopy, X-ray crystallography and complementary biochemistry.