Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.
MRC Toxicology Unit, University of Cambridge, Cambridge CB2 1QR, UK.
Nucleic Acids Res. 2022 Nov 11;50(20):11895-11915. doi: 10.1093/nar/gkac1048.
We previously identified RBPMS as a master regulator of alternative splicing in differentiated smooth muscle cells (SMCs). RBPMS is transcriptionally downregulated during SMC dedifferentiation, but we hypothesized that RBPMS protein activity might be acutely downregulated by post-translational modifications. Publicly available phosphoproteomic datasets reveal that Thr113 and Thr118 immediately adjacent to the RRM domain are commonly both phosphorylated. An RBPMS T113/118 phosphomimetic T/E mutant showed decreased splicing regulatory activity both in transfected cells and in a cell-free in vitro assay, while a non-phosphorylatable T/A mutant retained full activity. Loss of splicing activity was associated with a modest reduction in RNA affinity but significantly reduced RNA binding in nuclear extract. A lower degree of oligomerization of the T/E mutant might cause lower avidity of multivalent RNA binding. However, NMR analysis also revealed that the T113/118E peptide acts as an RNA mimic which can loop back and antagonize RNA-binding by the RRM domain. Finally, we identified ERK2 as the most likely kinase responsible for phosphorylation at Thr113 and Thr118. Collectively, our data identify a potential mechanism for rapid modulation of the SMC splicing program in response to external signals during the vascular injury response and atherogenesis.
我们之前发现 RBPMS 是分化平滑肌细胞（SMCs）中选择性剪接的主要调节因子。在 SMC 去分化过程中，RBPMS 的转录水平下调，但我们假设 RBPMS 蛋白活性可能会被翻译后修饰急性下调。公开的磷酸化蛋白质组数据集显示，紧邻 RRM 结构域的 Thr113 和 Thr118 通常同时被磷酸化。RBPMS T113/118 磷酸模拟 T/E 突变体在转染细胞和无细胞体外测定中表现出降低的剪接调节活性，而不可磷酸化的 T/A 突变体保留了全部活性。剪接活性的丧失与 RNA 亲和力的适度降低有关，但在核提取物中 RNA 结合显著减少。T/E 突变体的低聚化程度较低可能导致多价 RNA 结合的亲和力降低。然而，NMR 分析还表明，T113/118E 肽可充当 RNA 模拟物，可回折并拮抗 RRM 结构域的 RNA 结合。最后，我们确定 ERK2 是 Thr113 和 Thr118 磷酸化的最可能激酶。总之，我们的数据确定了一种潜在的机制，用于在血管损伤反应和动脉粥样硬化形成过程中快速调节 SMC 剪接程序以响应外部信号。
