支架附着因子B(SAFB)是一种保守的RNA结合蛋白,对哺乳动物的早期发育至关重要。然而,SAFB在小鼠胚胎干细胞(ESC)中的功能尚未得到表征。使用RNA免疫沉淀,然后是RNA-seq(RIP-seq),我们检测了野生型和SAFB/SAFB2双敲除ESCs中与SAFB相关的RNA。SAFB主要通过富含嘌呤的基序与蛋白质编码基因的内含子相关。在SAFB关联中最富集的转录物是lncRNAMalat1,其在其5'末端也含有富含嘌呤的区域。SAFB/SAFB2基因敲除导致约1,000个与多个生物过程相关的基因的差异表达。包括细胞凋亡,细胞分裂,和细胞迁移。SAFB/SAFB2的敲除还导致一组基因中的剪接变化,这些基因在很大程度上不同于表现出表达水平变化的基因。表达水平受SAFB正调控的许多基因的剪接和新生转录本也与高水平的SAFB相关。这意味着SAFB结合促进了它们的表达。将SAFB重新引入到双敲除细胞中,使基因表达恢复到野生型水平,在剪接和新生转录本水平上再次观察到的效果。蛋白质组学分析显示,SAFB相互作用者中核斑点相关蛋白和含RS结构域蛋白的显着富集。在SAFB/2敲除的ESC中Xist和Polycomb功能都没有显著改变。我们的发现表明,在ESC的其他潜在功能中,SAFB通过其结合新生RNA的能力促进某些基因的表达。
Scaffold attachment factor B (SAFB) is a conserved RNA-binding protein that is essential for early mammalian development. However, the functions of SAFB in mouse embryonic stem cells (ESCs) have not been characterized. Using RNA immunoprecipitation followed by RNA-seq (RIP-seq), we examined the RNAs associated with SAFB in wild-type and SAFB/SAFB2 double-knockout ESCs. SAFB predominantly associated with introns of protein-coding genes through purine-rich motifs. The transcript most enriched in SAFB association was the lncRNA Malat1, which also contains a purine-rich region in its 5\' end. Knockout of SAFB/SAFB2 led to differential expression of approximately 1000 genes associated with multiple biological processes, including apoptosis, cell division, and cell migration. Knockout of SAFB/SAFB2 also led to splicing changes in a set of genes that were largely distinct from those that exhibited changes in expression level. The spliced and nascent transcripts of many genes whose expression levels were positively regulated by SAFB also associated with high levels of SAFB, implying that SAFB binding promotes their expression. Reintroduction of SAFB into double-knockout cells restored gene expression toward wild-type levels, an effect again observable at the level of spliced and nascent transcripts. Proteomics analysis revealed a significant enrichment of nuclear speckle-associated and RS domain-containing proteins among SAFB interactors. Neither Xist nor Polycomb functions were dramatically altered in SAFB/2 knockout ESCs. Our findings suggest that among other potential functions in ESCs, SAFB promotes the expression of certain genes through its ability to bind nascent RNA.