PRMT1通过精氨酸甲基化作用修饰剪接因子SF2／ASF

Protein arginine methyltransferase 1 methylates SF2/ASF at arginine

目的:探讨蛋白质精氨酸甲基转移酶1(protein arginine methyltransferase1,PRMT1)对剪接因子SF2/ASF(spli-cing factor 2/alternative splicing factor)的甲基化修饰位点。方法:构建SF2/ASF野生型和Arg93/97/109突变体质粒,在体外表达和纯化GST标签的PRMT1、SF2/ASF及其Arg突变体融合蛋白,以甲基化活性实验检测PRMT1对SF2/ASF的甲基化作用及其甲基化修饰位点,以免疫荧光实验观察甲基化修饰对SF2/ASF亚细胞定位的影响。结果:PRMT1对SF2/ASF有明显的甲基化修饰作用;当Arg93/97/109突变为赖氨酸后,PRMT1对SF2/ASF突变体的甲基化修饰程度明显降低,其中Arg97突变后SF2/ASF甲基化程度减弱最明显。甲基化修饰不影响SF2/ASF的亚细胞定位。结论:发现SF2/ASF是PRMT1新的底物蛋白,Arg93/97/109均为PRMT1的甲基化修饰位点,其中Arg97是主要修饰位点;PRMT1对于SF2/ASF的甲基化修饰并不改变后者细胞内的定位。

Objective：To investigate the arginine （Arg） sites in splicing factor 2/aheruative splicing factor （SF2/ ASF） methylated by protein arginine methyhransferase 1 （PRMT1）. Methods： Wild-type and Arg93, Arg97, Argl09 mutant SF2/ASF plasmids were constructed, and GST-PRMT1, GST-SF2/ASF and arginine mutant GST-SF2/ASF fusion proteins were induced and purified. Methylation activity of PRMT1 on wild-type or mutant SF2/ASF protein and methyla- ted sites of SF2/ASF were examined by methylation assay. The effect of SF2/ASF methylation on its subcellular localization was analyzed by immunofluorescence assay. Results： PRMT1 induced methylation of SF2/ASF at arginine, and PRMT1 did not methylate SF2/ASF when SF2/ASF was mutant at Arg93, Arg97 or Argl09, with Arg97 mutation showing the most profound inhibitory effect. Methylation of SF2/ASF did not affect its subcellular localization. Conclusion： SF2/ ASF is a newly identified substrate of PRMT1 ; Arg93, Arg97 and Arg 109 are the three methylation sites in SF2/ASF, and Arg97 is the main methylation site. Methylation of SF2/ASF does not affect its subcellular localization.