RBM20 phosphorylation and its role in nucleocytoplasmic transport and cardiac pathogenesis.
RBM20 phosphorylation and its role in nucleocytoplasmic transport and cardiac pathogenesis.
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DOI：
10.1096/fj.202101811rr
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发表时间：
2022-05
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剪接因子中的精氨酸-丝氨酸（RS）结构域对于前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剪接控制和细胞核质转运具有重要意义。RNA结合基序20（RBM 20）是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被表征。使用中-下质谱法，我们鉴定了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结构域的RSRSP片段中。S638和S640上的突变调节剪接，促进核质转运和蛋白质-RNA缩合物。S638和S640的拟磷酸化突变表明，磷酸化不是体外RBM 20核质转运和浓缩的主要原因。我们生成了S637 A敲入（KI）小鼠模型（Rbm 20 S637 A），并观察到RBM 20磷酸化降低。KI小鼠表现出异常基因剪接、蛋白质缩合物和扩张型心肌病（DCM）样表型。转录组学分析表明，KI小鼠改变了涉及心脏功能障碍，蛋白质定位和冷凝的基因的表达和剪接。我们在体外的数据表明，磷酸化不是一个直接的原因，核质运输和蛋白质凝聚。随后，体内结果显示RBM 20突变导致心脏发病。然而，磷酸化在体内的作用需要进一步研究。
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 (Rbm20S637A) 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.