SF3B1 K700E Mutation Induces Genome-Wide Enhancement of Transcriptional Pause Release in ES Cells [RNA-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 biological conditions by RNA-seq; 3 biological replicate sets of SF3B1 K700E Mutant and SF3B1 WT libraries. 3 technical replicates per biological replicate.

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）剪接过程中发挥核心作用。本研究利用CRISPR-Cas9技术，在人类胚胎干细胞（human embryonic stem cells, hESC）中构建了最常见的SF3B1突变——赖氨酸700位谷氨酸杂合取代（K700E）的杂合克隆株，以此探究该突变的生物学效应。研究观察到，携带SF3B1K700E突变的hESC中，多个与造血功能相关的关键转录调控因子以及广泛的免疫基因均出现上调表达。尽管携带SF3B1K700E突变的hESC与骨髓增生异常综合征（myelodysplastic syndrome, MDS）细胞存在转录组差异，但两类细胞中均鉴定出若干共有的基因表达程序，且该现象与剪接改变无关。为进一步阐明SF3B1K700E突变hESC中基因表达失调的分子机制，我们对突变细胞开展了精确运行测序（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能参与介导SF3B1突变表型的下游候选基因，为解析U2 snRNP通过RNA聚合酶II调控转录的机制提供了新的研究视角。整体实验设计：通过RNA测序（RNA-seq）检测6组生物学样本，包含SF3B1 K700E突变型与野生型（wild type, WT）文库各3套生物学重复，每套生物学重复设置3次技术重复。