RBPMS and RBPMS2 Cooperate to Safeguard Cardiac Splicing P0

RBPMS and RBPMS2 share a high amino acid sequence similarity and are interchangeable in in vitro splicing assays. However, it remains unclear whether RBPMS and RBPMS2 play similar or distinct function during heart development in vivo. To address this question, we constructed Rbpms and Rbpms2 floxed mice with an established CRISPR-Cas9 method, and validated the efficacy of Rbpms or Rbpms2 floxed alleles in ablating RBPMS or RBPMS2 proteins by crossing them with germline-expressing Sox2-Cre. Transcriptome were abtained by RNA seq to investigate the cardiac function of RBPMS and RBPMS2. We performed RNA sequencing on RbpmscmKO, Rbpms2cmKO, and Rbpms/Rbpms2dcmKO hearts along with their littermate controls. In this study, we generated cardiomyocyte-specific knockout (KO) mice for Rbpms, Rbpms2, and both genes combined (double KO). The experimental groups included Rbpms single KO, Rbpms2 single KO, and Rbpms/Rbpms2 double KO mice, which were analyzed to assess cardiac phenotypes. RNA sequencing was conducted on P0 heart tissue from each KO group to evaluate changes in gene expression and splicing patterns. In silico analyses were used to dissect the mechanisms behind the distinct and overlapping roles of RBPMS and RBPMS2 in splicing regulation during heart development. The study focused on identifying the effects of RBPMS and RBPMS2 on sarcomere assembly and the regulation of cardiac-specific splicing programs, as well as determining the impact of their binding locations on pre-mRNA.

RBPMS与RBPMS2具备高度相似的氨基酸序列，在体外剪接实验中可实现功能互换。但目前仍不清楚二者在体内心脏发育过程中，是发挥相似功能还是具有截然不同的作用。为解答这一科学问题，本研究依托成熟的CRISPR-Cas9技术构建了Rbpms与Rbpms2 floxed小鼠（floxed mice），并通过将其与生殖系表达Sox2-Cre的小鼠杂交，验证了Rbpms或Rbpms2的floxed等位基因（floxed alleles）可有效敲除RBPMS或RBPMS2蛋白。本研究通过RNA测序（RNA-seq）获取转录组数据，以探究RBPMS与RBPMS2的心脏相关功能。我们对Rbpms心肌细胞特异性敲除（cardiomyocyte-specific knockout, cmKO）小鼠（RbpmscmKO）、Rbpms2心肌细胞特异性敲除小鼠（Rbpms2cmKO）、Rbpms与Rbpms2双基因心肌细胞特异性敲除小鼠（Rbpms/Rbpms2dcmKO）的心脏组织，及其同窝野生型对照样本进行了RNA测序。本研究成功构建了针对Rbpms、Rbpms2以及双基因联合敲除的心肌细胞特异性敲除（knockout, KO）小鼠模型。实验组涵盖Rbpms单基因敲除小鼠、Rbpms2单基因敲除小鼠与Rbpms/Rbpms2双基因敲除小鼠，通过对上述模型开展分析以评估其心脏表型。我们对各敲除组小鼠出生后第0天（postnatal day 0, P0）的心脏组织实施RNA测序，以评估基因表达与剪接模式的变化情况。本研究采用计算机模拟分析（in silico analyses）手段，解析RBPMS与RBPMS2在心脏发育过程中剪接调控环节的独特功能与重叠功能背后的分子机制。本研究重点聚焦于明确RBPMS与RBPMS2对肌节组装以及心脏特异性剪接程序的调控作用，同时探究二者的结合位点对前体mRNA（pre-mRNA）的影响效应。