Intron branchpoint (BP) recognition by the U2 snRNP is a critical step of splicing, vulnerable to recurrent cancer mutations and bacterial natural product inhibitors. The BP binds a conserved pocket in the SF3B1 (human) or Hsh155 (yeast) U2 snRNP protein. Amino acids that line this pocket affect the binding of splicing inhibitors like Pladienolide-B (Plad-B), such that organisms differ in their sensitivity. To study the mechanism of splicing inhibitor action in a simplified system, we modified the naturally Plad-B resistant yeast Saccharomyces cerevisiae by changing 14 amino acids in the Hsh155 BP pocket to those from human. This humanized yeast grows normally, and splicing is largely unaffected by the mutation. Splicing is inhibited within minutes after the addition of Plad-B, and different introns appear inhibited to different extents. Intron-specific inhibition differences are also observed during cotranscriptional splicing in Plad-B using single-molecule intron tracking to minimize gene-specific transcription and decay rates that cloud estimates of inhibition by standard RNA-seq. Comparison of Plad-B intron sensitivities to those of the structurally distinct inhibitor Thailanstatin-A reveals intron-specific differences in sensitivity to different compounds. This work exposes a complex relationship between the binding of different members of this class of inhibitors to the spliceosome and intron-specific rates of BP recognition and catalysis. Introns with variant BP sequences seem particularly sensitive, echoing observations from mammalian cells, where monitoring individual introns is complicated by multi-intron gene architecture and alternative splicing. The compact yeast system may hasten the characterization of splicing inhibitors, accelerating improvements in selectivity and therapeutic efficacy.

中文翻译：

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人源化酵母菌株中各个内含子对剪接抑制剂的反应存在广泛差异

U2 snRNP 的内含子分支点 (BP) 识别是剪接的关键步骤，容易受到复发性癌症突变和细菌天然产物抑制剂的影响。BP 结合 SF3B1（人）或 Hsh155（酵母）U2 snRNP 蛋白中的保守口袋。该口袋中的氨基酸会影响 Pladienolide-B (Plad-B) 等剪接抑制剂的结合，从而使生物体的敏感性有所不同。为了研究简化系统中剪接抑制剂的作用机制，我们通过将 Hsh155 BP 口袋中的 14 个氨基酸更改为来自人类的氨基酸，对天然 Plad-B 抗性酵母酿酒酵母进行了改造。这种人源化酵母生长正常，剪接基本上不受突变的影响。添加Plad-B后几分钟内剪接就被抑制，并且不同的内含子出现不同程度的抑制。在 Plad-B 的共转录剪接过程中，使用单分子内含子追踪也观察到了内含子特异性抑制差异，以最大限度地减少基因特异性转录和衰减率，从而使标准 RNA-seq 的抑制估计变得模糊。Plad-B 内含子敏感性与结构不同的抑制剂 Thailanstatin-A 的比较揭示了对不同化合物敏感性的内含子特异性差异。这项工作揭示了此类抑制剂的不同成员与剪接体的结合以及 BP 识别和催化的内含子特异性速率之间的复杂关系。具有变异 BP 序列的内含子似乎特别敏感，这与哺乳动物细胞的观察结果相呼应，在哺乳动物细胞中，监测单个内含子因多内含子基因结构和选择性剪接而变得复杂。紧凑的酵母系统可以加速剪接抑制剂的表征，加速选择性和治疗功效的提高。