Assessment of transcript isoforms in Esrp1Triaka versus wild-type primary colonic epithelial cells. RNA sequencing of intestinal epithelial cells from Esrp1Triaka mutant mice

Alternative mRNA splicing is a process to increase the biodiversity of proteins that occurs in the majority of higher eukaryotic pre-mRNA. Altered mRNA splicing has been associated with several human diseases. Epithelial splicing regulatory protein 1 (ESRP1) was identified in a cDNA expression screen for factors promoting the epithelial pattern of FGFR2 splicing. ESRP1 was also found to regulate the alternative splicing of CD44 and other genes. Esrp1 is epithelial cell-restricted and is highly expressed in the murine large intestine. Here, we used a mouse mutant of Esrp1 called Triaka to investigate the function of ESRP1 protein in the intestine. Triaka mice are described in details under the following link: https://mutagenetix.utsouthwestern.edu/phenotypic/phenotypic_rec.cfm?pk=354. We first performed in vitro experiments using an exon trap vector based on luciferase expression, which indicated that the Esrp1Triaka allele leads to reduced mRNA splicing activity of the ESRP1 protein. In addition, RNA sequencing analysis showed altered patterns of transcript isoforms in Esrp1Triaka versus wild-type primary colonic epithelial cells. This was associated with impaired intestinal barrier integrity during homeostasis, increased susceptibility to experimental colitis and reduced colonic epithelial wound-healing capacity in Triaka mice. Together, these data indicate a central role of ESRP1 for intestinal barrier integrity.

可变mRNA剪接（alternative mRNA splicing）是一种提升蛋白质多样性的生物学过程，广泛存在于绝大多数高等真核生物的前体mRNA（pre-mRNA）中。异常mRNA剪接与多种人类疾病密切相关。上皮剪接调控蛋白1（Epithelial splicing regulatory protein 1，ESRP1）最初在一项靶向促进FGFR2剪接上皮特异性模式的因子的互补DNA（cDNA）表达筛选实验中被鉴定得到。后续研究还发现，ESRP1可调控CD44及其他基因的可变剪接。Esrp1基因仅在上皮细胞中特异性表达，并在小鼠大肠中高度富集。本研究使用一种名为Triaka的Esrp1基因突变小鼠模型，探究ESRP1蛋白在肠道中的功能。有关Triaka小鼠的详细资料可通过以下链接查阅：https://mutagenetix.utsouthwestern.edu/phenotypic/phenotypic_rec.cfm?pk=354。本研究首先采用基于荧光素酶表达的外显子捕获载体开展体外实验，结果显示Esrp1Triaka等位基因可降低ESRP1蛋白的mRNA剪接活性。此外，RNA测序（RNA sequencing）分析显示，与野生型原代结肠上皮细胞相比，Esrp1Triaka突变细胞的转录本异构体表达模式发生了显著改变。该突变与Triaka小鼠在稳态条件下的肠道屏障完整性受损、实验性结肠炎易感性升高以及结肠上皮伤口愈合能力下降密切相关。综上，上述实验数据表明ESRP1在维持肠道屏障完整性中发挥核心作用。