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

前mRNA剪接发生在剪接体上，剪接体是一种大型蛋白质-RNA复合物，其去除非编码内含子并将编码外显子连接成成熟RNA。剪接体核糖核蛋白U2（snRNP）位于前体mRNA的分支位点区域，是剪接调控中的关键分子。U2 snRNP的SF 3 B复合物的突变在各种形式的癌症中有很高的发病率，并通过目前尚不清楚的机制导致异常剪接。此外，几种具有抗肿瘤特性的剪接调节剂，如普拉地辛B、herboxidiene和spliceostatin A，结合SF 3 B复合物并抑制正常的分支位点使用，诱导内含子保留和外显子跳跃。尽管经过十年的研究，调节剂的结合位点及其作用机制仍然不清楚。我们的目标是在理解致癌突变和具有治疗潜力的抗肿瘤化合物调节剪接调节的机制方面取得突破。具体来说，我们的目标是工程spliceosomal复合物和SF 3b的变体，捕获剪接调节下的病理和药物诱导的条件下，并通过电子冷冻显微镜，X射线晶体学和互补的生物化学表征其三维结构。

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.