Hotspot mutations in the spliceosome gene SF3B1 are reported in ∼20% of uveal melanomas. SF3B1 is involved in 3'-splice site (3'ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear. Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1(R625/K666) mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3'ss. Modelling the differential junctions in SF3B1(WT) and SF3B1(R625/K666) cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3'ss-sequence context. SF3B1(WT) knockdown or overexpression do not reproduce the SF3B1(R625/K666) splice pattern, qualifying SF3B1(R625/K666) as change-of-function mutants. Mutagenesis of predicted branchpoints reveals that the SF3B1(R625/K666)-promoted splice pattern is a direct result of alternative branchpoint usage. Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease.

中文翻译：

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癌症相关的 SF3B1 突变通过促进替代分支点的使用来影响可变剪接。

在约 20% 的葡萄膜黑色素瘤中报道了剪接体基因 SF3B1 的热点突变。SF3B1 在 RNA 剪接过程中参与 3'-剪接位点 (3's) 识别；然而，其突变的分子机制仍不清楚。在这里，我们使用对葡萄膜黑色素瘤的 RNA-Seq 分析表明，SF3B1(R625/K666) 突变导致连接子集的剪接失调，主要是通过使用替代 3'ss。对 SF3B1(WT) 和 SF3B1(R625/K666) 细胞系中的差异连接进行建模表明，失调的剪接模式严格取决于 SF3B1 状态和 3'ss 序列上下文。SF3B1(WT) 敲低或过表达不会复制 SF3B1(R625/K666) 剪接模式，将 SF3B1(R625/K666) 限定为功能改变突变体。预测分支点的诱变表明，SF3B1(R625/K666) 促进的剪接模式是替代分支点使用的直接结果。总而言之，这项研究提供了对突变 SF3B1 在癌症中诱导的剪接改变的机制的更好理解，并揭示了替代分支点在疾病中的作用。