SF3B1 K700E Mutation Induces Genome-Wide Enhancement of Transcriptional Pause Release in ES Cells [PRO-seq]

SF3B1, a critical component of the U2 snRNP splicing factor, is frequently mutated in cancer and plays a crucial role in pre-mRNA splicing. We investigated the effects of the most common SF3B1 mutation, heterozygous substitution of Lysine 700 to Glutamate (K700E), in human embryonic stem (hESC) cells using CRISPR-Cas9 to generate heterozygous SF3B1K700E clones. We observed the upregulation of several key transcription regulators associated with hematopoiesis and a broad range of immune genes in SF3B1K700E hESCs. Despite transcriptional differences between hESC and myelodysplastic syndrome (MDS) cells harboring the SF3B1K700E mutation, several common gene programs were identified in both cell types, independent of splicing alterations. To further elucidate the molecular mechanisms underlying dysregulated gene expression in SF3B1K700E hESCs, we performed Precision Run-On sequencing (PRO-seq) in SF3B1K700E hESCs. These analyses revealed alterations in RNA polymerase II (Pol II) elongation properties induced by the SF3B1K700E mutation. Specifically, a general increase in pause release was noted in SF3B1K700E hESCs. This study identifies several downstream candidate genes that could contribute to the SF3B1 mutated phenotype, shedding light on the impact of the U2 snRNP on transcription by Pol II. Overall design: Examination of 6 samples by PRO-seq; 3 biological replicate sets of SF3B1 K700E Mutant and SF3B1 WT libraries.

SF3B1是U2小核糖核蛋白（U2 snRNP）剪接因子的关键组成部分，在癌症中频发突变，且在前体mRNA（pre-mRNA）剪接过程中发挥至关重要的作用。我们采用CRISPR-Cas9基因编辑系统（CRISPR-Cas9）构建携带SF3B1最常见突变——赖氨酸700位谷氨酸杂合替换（K700E）的人胚胎干细胞（human embryonic stem cell, hESC）克隆，进而探究该突变在人胚胎干细胞中的生物学效应。我们观察到，在SF3B1K700E型人胚胎干细胞中，多种与造血作用相关的关键转录调控因子以及广泛的免疫基因均出现表达上调。尽管携带SF3B1K700E突变的人胚胎干细胞与骨髓增生异常综合征（myelodysplastic syndrome, MDS）细胞之间存在转录组差异，但两类细胞中均鉴定出若干共通的基因表达程序，且该现象与剪接改变无关。为进一步阐明SF3B1K700E型人胚胎干细胞中基因表达失调的分子机制，我们对该类细胞开展了精准延伸测序（Precision Run-On sequencing, PRO-seq）分析。结果显示，SF3B1K700E突变可改变RNA聚合酶II（RNA polymerase II, Pol II）的延伸特性，具体表现为该突变型细胞中RNA聚合酶II的暂停释放过程普遍增强。本研究鉴定出若干可参与介导SF3B1突变表型的下游候选基因，为揭示U2小核糖核蛋白通过RNA聚合酶II调控转录的机制提供了新的见解。实验设计：通过PRO-seq对6份样本进行检测，包含3组生物学重复的SF3B1 K700E突变型文库与野生型（wild type, WT）文库。