RNA-binding motif protein 20 (RBM20) is a cardiac splice regulator that adapts cardiac filling via its diverse substrates—including the sarcomeric protein titin. The molecular basis and regulation of RBM20-dependent exon exclusion are largely unknown. In tissue culture experiments, we show that the combination of RNA recognition motif (RRM) and C-terminus is necessary and sufficient for RBM20 activity, indicating an important function of the ZnF2 domain in splicing repression. Using splice reporter and in vitro binding assays targeting titin exons 241–243, we identified a minimal genomic segment that is necessary for RBM20-mediated splicing repression of the alternative exon. Here, RBM20 binds the cluster containing most RBM20 binding motifs through its RRM domain and represses the upstream and downstream introns. For subsequent exon exclusion, specific regions upstream, downstream and within the alternative exon 242 are required. Regulation of exon exclusion involves PTB4 as a novel titin splice regulator, which counteracts RBM20 repressor activity in HEK293 cells. Together, these mechanistic insights into the regulation and action of RBM20 and PTB4 provide a basis for the future development of RBM20 modulators that adapt titin elasticity in cardiac disease.

中文翻译：

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RBM20和新型的Titin剪接调节剂PTB4排除Titin外显子的分子基础

RNA结合基序蛋白20（RBM20）是一种心脏剪接调节剂，可通过其多种底物（包括肌节蛋白滴定蛋白）适应心脏充盈。RBM20依赖的外显子排斥的分子基础和调节是很大程度上未知。在组织培养实验中，我们显示RNA识别基序（RRM）和C端的组合对于RBM20活性是必要且充分的，表明ZnF2域在剪接抑制中具有重要作用。使用剪接报告物和体外结合针对titin外显子241-243的结合测定，我们确定了最小基因组片段，这是RBM20介导的替代外显子剪接抑制所必需的。在这里，RBM20通过其RRM结构域结合包含大多数RBM20结合基序的簇，并抑制上游和下游内含子。对于随后的外显子排除，需要替代外显子242上游，下游和内部的特定区域。外显子排阻的调节涉及PTB4作为新型的titin剪接调节剂，它抵消了HEK293细胞中的RBM20阻遏物活性。综上所述，这些对RBM20和PTB4调控和作用的机械性见解为适应心脏疾病中钛蛋白弹性的RBM20调节剂的未来发展提供了基础。