目的:探讨蛋白质精氨酸甲基转移酶1 (protein arginine methyltransferase 1，PRMT1)对剪接因子SF2/ASF (splicing 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/alternative splicing factor (SF2/ASF) methylated by protein arginine methyltransferase 1 (PRMT1). Methods: Wildtype and Arg93, Arg97, Arg109 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 methylated 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 Arg109, 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.

国家863计划现代医学技术重大资助项目（No.2006AA02A405）；国家863计划现代医学技术专题项目（No. 2006AA02Z4Z2）