Aims Differentiated vascular smooth muscle cells (VSMCs) express a unique network of mRNA isoforms via smooth muscle–specific alternative pre-mRNA splicing (SM-AS) in functionally critical genes, including those comprising the contractile machinery. We previously described RNA Binding Protein with Multiple Splicing (RBPMS) as a potent driver of differentiated SM-AS in the rat PAC1 VSMC cell line. What is unknown is how RBPMS affects VSMC phenotype and behaviour. Here, we aimed to dissect the role of RBPMS in SM-AS in human cells and determine the impact on VSMC phenotypic properties. Methods and results We used human embryonic stem cell–derived VSMCs (hESC-VSMCs) as our platform. hESC-VSMCs are inherently immature, and we found that they display only partially differentiated SM-AS patterns while RBPMS protein levels are low. We found that RBPMS over-expression induces SM-AS patterns in hESC-VSMCs akin to the contractile tissue VSMC splicing patterns. We present in silico and experimental findings that support RBPMS’ splicing activity as mediated through direct binding and via functional cooperativity with splicing factor RBFOX2 on a significant subset of targets. We also demonstrate that RBPMS can alter the motility and the proliferative properties of hESC-VSMCs to mimic a more differentiated state. Conclusion Overall, this study emphasizes a critical role for RBPMS in establishing the contractile phenotype splicing programme of human VSMCs.

中文翻译：

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具有多重剪接的 RNA 结合蛋白 （RBPMS） 促进人胚胎干细胞来源的血管平滑肌细胞中的收缩表型剪接


目的 分化的血管平滑肌细胞 （VSMC） 通过功能关键基因（包括构成收缩机制的基因）中的平滑肌特异性选择性前体 mRNA 剪接 （SM-AS） 表达独特的 mRNA 亚型网络。我们之前将多重剪接的 RNA 结合蛋白 （RBPMS） 描述为大鼠 PAC1 VSMC 细胞系中分化 SM-AS 的有效驱动因素。尚不清楚 RBPMS 如何影响 VSMC 表型和行为。在这里，我们旨在剖析 RBPMS 在人类细胞 SM-AS 中的作用，并确定对 VSMC 表型特性的影响。方法和结果我们使用人胚胎干细胞衍生的 VSMC （hESC-VSMCs） 作为我们的平台。hESC-VSMCs 本质上是不成熟的，我们发现它们只显示部分分化的 SM-AS 模式，而 RBPMS 蛋白水平较低。我们发现 RBPMS 过表达在 hESC-VSMC 中诱导 SM-AS 模式，类似于收缩组织 VSMC 剪接模式。我们以计算机和实验结果展示了支持 RBPMS 的剪接活性，该活性是通过直接结合和通过与剪接因子 RBFOX2 在重要靶标子集上的功能协同性介导的。我们还证明 RBPMS 可以改变 hESC-VSMC 的运动和增殖特性以模拟分化程度更高的状态。结论 总体而言，本研究强调了 RBPMS 在建立人类 VSMC 的收缩表型剪接程序中的关键作用。