Department of Animal and Dairy Sciences, University of Wisconsin-Madison, Wisconsin, USA.
Department of Animal Science, University of Wyoming, Laramie, Wyoming, USA.
FASEB J. 2022 May;36(5):e22302. doi: 10.1096/fj.202101811RR.
Arginine-serine (RS) domain(s) in splicing factors are critical for protein-protein interaction in pre-mRNA splicing. Phosphorylation of RS domain is important for splicing control and nucleocytoplasmic transport in the cell. RNA-binding motif 20 (RBM20) is a splicing factor primarily expressed in the heart. A previous study using phospho-antibody against RS domain showed that RS domain can be phosphorylated. However, its actual phosphorylation sites and function have not been characterized. Using middle-down mass spectrometry, we identified 16 phosphorylation sites, two of which (S638 and S640 in rats, or S637 and S639 in mice) were located in the RSRSP stretch in the RS domain. Mutations on S638 and S640 regulated splicing, promoted nucleocytoplasmic transport and protein-RNA condensates. Phosphomimetic mutations on S638 and S640 indicated that phosphorylation was not the major cause for RBM20 nucleocytoplasmic transport and condensation in vitro. We generated a S637A knock-in (KI) mouse model (Rbm20 ) and observed the reduced RBM20 phosphorylation. The KI mice exhibited aberrant gene splicing, protein condensates, and a dilated cardiomyopathy (DCM)-like phenotype. Transcriptomic profiling demonstrated that KI mice had altered expression and splicing of genes involving cardiac dysfunction, protein localization, and condensation. Our in vitro data showed that phosphorylation was not a direct cause for nucleocytoplasmic transport and protein condensation. Subsequently, the in vivo results reveal that RBM20 mutations led to cardiac pathogenesis. However, the role of phosphorylation in vivo needs further investigation.
精氨酸-丝氨酸（RS）结构域在剪接因子中对于前体 mRNA 剪接中的蛋白质-蛋白质相互作用至关重要。RS 结构域的磷酸化对于细胞中的剪接调控和核质转运非常重要。RNA 结合基序 20（RBM20）是一种主要在心脏中表达的剪接因子。先前使用针对 RS 结构域的磷酸化抗体的研究表明，RS 结构域可以被磷酸化。然而，其实际的磷酸化位点和功能尚未得到表征。使用中向下质谱法，我们鉴定出 16 个磷酸化位点，其中两个（大鼠中的 S638 和 S640，或小鼠中的 S637 和 S639）位于 RS 结构域中的 RSRSP 延伸区。S638 和 S640 上的突变调节剪接，促进核质转运和蛋白质-RNA 凝聚。S638 和 S640 的磷酸模拟突变表明，磷酸化不是 RBM20 体外核质转运和凝聚的主要原因。我们生成了 S637A 敲入（KI）小鼠模型（Rbm20 ），并观察到 RBM20 磷酸化减少。KI 小鼠表现出异常的基因剪接、蛋白质凝聚和扩张型心肌病（DCM）样表型。转录组谱分析表明，KI 小鼠的基因表达和剪接发生改变，涉及心脏功能障碍、蛋白质定位和凝聚。我们的体外数据表明，磷酸化不是核质转运和蛋白质凝聚的直接原因。随后，体内结果表明 RBM20 突变导致心脏发病机制。然而，体内磷酸化的作用需要进一步研究。
