Wu Tongbin, Chen Zeyu, Zhang Zengming, Zhou Xiaohai, Gu Yusu, Dinenno Frank A, Chen Ju
bioRxiv. 2024 Nov 9:2024.11.07.622565. doi: 10.1101/2024.11.07.622565.
Mutations in cardiac splicing factors (SFs) cause cardiomyopathy and congenital heart disease, underscoring the critical role of SFs in cardiac development and disease. Cardiac SFs are implicated to cooperatively regulate the splicing of essential cardiac genes, but the functional importance of their collaboration remains unclear. RNA Binding Protein with Multiple Splicing (RBPMS) and RBPMS2 are SFs involved in heart development and exhibit similar splicing regulatory activities , but it is unknown whether they cooperate to regulate splicing .
and single or double cardiomyocyte (CM)-specific knockout (KO) mice were generated and analyzed for cardiac phenotypes. RNA sequencing was performed to assess gene expression and splicing changes in single and double KOs. analyses were used to dissect the mechanisms underlying distinct and overlapping roles of RBPMS and RBPMS2 in heart development.
Mice lacking both RBPMS and RBPMS2 in CMs died before embryonic day 13.5 and developed sarcomere disarray, whereas or single CM-specific KO mice had normal sarcomere assembly and survived to adulthood. Defective sarcomere assembly is likely owing to the widespread mis-splicing of genes essential for cardiac contraction in double KO mice, underscoring the overlapping role of RBPMS and RBPMS2 in splicing regulation. Mechanistically, we found RBPMS and RBPMS collectively promote cardiac splicing program while repressing non-cardiac splicing programs. Moreover, RNA splicing maps suggested that the binding location of RBPMS and RBPMS2 on pre-mRNA dictates whether they function as splicing activators or repressors. Lastly, the requirement for RBPMS and/or RBPMS2 for splicing regulation arises from intrinsic features of the target exons.
Our results demonstrate that RBPMS and RBPMS2 work in concert to safeguard the splicing of genes essential for cardiac contraction, highlighting the importance of SF collaboration in maintaining cardiac splicing signature, which should be taken into consideration when devising future therapeutic approaches through modulating the activity of SFs.
Mutations in cardiac splicing factors (SFs) cause cardiomyopathy and congenital heart disease, and the splicing of cardiac genes is regulated by multiple SFs. However, the functional importance of the collaboration among specific cardiac SFs is unknown.RBPMS has emerged as a cardiac SF for sarcomere genes but is not required for sarcomere assembly. RBPMS2 can substitute RBPMS in splicing assays, yet its role in mammalian cardiomyocytes (CMs) remains unclear. RBPMS and RBPMS2 have both distinct and overlapping roles in CMs.RBPMS and RBPMS2 collectively contribute to the maintenance of cardiac splicing program, which is essential for sarcomere assembly and embryonic survival.RNA splicing map of RBPMS and RBPMS2 reveals that they can function either as splicing activators or repressors, depending on their binding locations on pre-mRNA. This study provides compelling evidence of cooperation between cardiac splicing factors during heart development, which, to our knowledge, has not been demonstrated . and CM-specific double KO mice die in utero and exhibit sarcomere disarray, whereas single KO mice survive to adulthood with normal sarcomere structure but manifest distinct cardiac phenotypes, suggesting RBPMS and RBPMS2 possess both distinct and overlapping functions in CMs. Although mis-splicing in cardiac genes can be seen in all three KOs, the splicing signature of double KO hearts drastically shifts towards non-cardiac tissues, including more prominent mis-splicing in genes related to cardiac contractile function. Our study further reveals that the splicing regulation of RBPMS and RBPMS2 has the characteristics of "positional effects", i.e., the binding location on pre-mRNA dictates whether they function as splicing activators or repressors; and the intrinsic features of the target exon determine the requirement for one or two RBPMS proteins for splicing regulation. Our study sheds light on the functional importance of cardiac SF cooperation in maintaining cardiac splicing signature during heart development.
心脏剪接因子（SFs）的突变会导致心肌病和先天性心脏病，这突出了剪接因子在心脏发育和疾病中的关键作用。心脏剪接因子被认为协同调节心脏必需基因的剪接，但其协作的功能重要性仍不清楚。具有多个剪接功能的RNA结合蛋白（RBPMS）和RBPMS2是参与心脏发育的剪接因子，表现出相似的剪接调节活性，但它们是否协同调节剪接尚不清楚。
构建并分析了RBPMS和RBPMS2单基因或双基因心肌细胞（CM）特异性敲除（KO）小鼠的心脏表型。进行RNA测序以评估单基因和双基因敲除小鼠中的基因表达和剪接变化。采用分析方法剖析RBPMS和RBPMS2在心脏发育中不同和重叠作用的潜在机制。
心肌细胞中同时缺乏RBPMS和RBPMS2的小鼠在胚胎第13.5天前死亡，并出现肌节紊乱，而RBPMS或RBPMS2单基因CM特异性敲除小鼠的肌节组装正常，可存活至成年。双基因敲除小鼠中肌节组装缺陷可能是由于心脏收缩必需基因的广泛错配剪接，这突出了RBPMS和RBPMS2在剪接调节中的重叠作用。从机制上讲，我们发现RBPMS和RBPMS2共同促进心脏剪接程序，同时抑制非心脏剪接程序。此外，RNA剪接图谱表明RBPMS和RBPMS2在pre-mRNA上的结合位置决定了它们是作为剪接激活剂还是抑制剂发挥作用。最后，剪接调节对RBPMS和/或RBPMS2的需求源于靶外显子的内在特征。
我们的结果表明，RBPMS和RBPMS2协同作用以保障心脏收缩必需基因的剪接，突出了剪接因子协作在维持心脏剪接特征中的重要性，在通过调节剪接因子活性设计未来治疗方法时应予以考虑。
心脏剪接因子（SFs）的突变会导致心肌病和先天性心脏病，心脏基因的剪接由多种剪接因子调节。然而，特定心脏剪接因子之间协作的功能重要性尚不清楚。RBPMS已成为肌节基因的心脏剪接因子，但不是肌节组装所必需的。RBPMS2在剪接试验中可以替代RBPMS，但其在哺乳动物心肌细胞（CMs）中的作用仍不清楚。RBPMS和RBPMS2在心肌细胞中具有不同和重叠的作用。RBPMS和RBPMS2共同有助于维持心脏剪接程序，这对肌节组装和胚胎存活至关重要。RBPMS和RBPMS2的RNA剪接图谱显示，它们可以作为剪接激活剂或抑制剂发挥作用，这取决于它们在pre-mRNA上的结合位置。本研究提供了令人信服的证据，证明心脏发育过程中心脏剪接因子之间的协作，据我们所知，此前尚未得到证实。RBPMS和RBPMS2双基因CM特异性敲除小鼠在子宫内死亡并表现出肌节紊乱，而单基因敲除小鼠存活至成年，肌节结构正常，但表现出不同的心脏表型，表明RBPMS和RBPMS2在心肌细胞中具有不同和重叠的功能。尽管在所有三种敲除小鼠中都可以看到心脏基因的错配剪接，但双基因敲除心脏的剪接特征急剧转向非心脏组织，包括与心脏收缩功能相关基因中更明显的错配剪接。我们的研究进一步揭示，RBPMS和RBPMS2的剪接调节具有“位置效应”的特征，即它们在pre-mRNA上的结合位置决定了它们是作为剪接激活剂还是抑制剂发挥作用；靶外显子的内在特征决定了剪接调节对一种或两种RBPMS蛋白的需求。我们的研究揭示了心脏剪接因子协作在心脏发育过程中维持心脏剪接特征的功能重要性。
