Knockout mice reveal an essential role for Epithelial splicing regulatory proteins (Esrps) in mammalian development and epithelial splicing in vivo

Tissue- and cell-type specific regulators of alternative splicing (AS) are an essential layer of posttranscriptional gene regulation necessary for normal cellular function, patterning, and development. Here we report the Epithelial splicing regulatory proteins (Esrps) are required for patterning of multiple organs, with loss of both paralogs, Esrp1 and Esrp2, resulting in increasingly severe phenotypes. Global profiling of the Esrp splicing regulatory network from total epidermis revealed varied splicing sensitivity of Esrp targets upon loss of Esrp1 or double knockout. This may explain the progressive phenotypes seen in Esrp knockout mice, and these mice provide a unique genetic tool to evaluate functional consequences of epithelial splicing events in vivo. RNA from purified total epidermis (basal keratinocyte layer to cornified layer) of E18.5 mouse embryos were harvested by Trizol extraction. (Esrp1+/+, Esrp2-/- (n=2), Esrp1-/-, Esrp2+/+ (n=3), Esrp1-/-, Esrp2+/- (n=2), and Esrp1-/-, Esrp2-/- (n=2). 1 ug of total RNA was used for for RNA-seq library preparation using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina). 100x2 bp paired-end RNA-seq reads were generated on a HiSeq 2000 sequencer.可变剪接（alternative splicing, AS）的组织与细胞类型特异性调控因子，是维持正常细胞功能、模式形成与个体发育所必需的转录后基因调控核心层级。本研究证实上皮剪接调控蛋白（Epithelial splicing regulatory proteins, Esrps）是多个器官模式形成所必需的调控因子；当两个旁系同源基因Esrp1与Esrp2均发生缺失时，会导致表型程度逐渐加重。通过对全表皮组织中Esrp剪接调控网络的全局谱分析，我们发现Esrp靶基因的剪接敏感性在Esrp1单基因缺失或双基因敲除时存在显著差异。这一发现或许可以解释Esrp敲除小鼠中观察到的进行性表型，且此类小鼠为在体内评估上皮剪接事件的功能后果提供了独特的遗传工具。我们采用Trizol法提取了E18.5小鼠胚胎纯化全表皮组织（从基底角质形成细胞层到角质层）的总RNA，样本基因型包括：Esrp1+/+、Esrp2-/-（n=2）；Esrp1-/-、Esrp2+/+（n=3）；Esrp1-/-、Esrp2+/-（n=2）；以及Esrp1-/-、Esrp2-/-（n=2）。取1 μg总RNA，使用TruSeq链特异性mRNA LT样本制备试剂盒（Illumina公司）进行RNA测序文库构建，并在HiSeq 2000测序仪上生成100×2 bp的双端RNA测序读段。