Passos Dario O, Quaresma Alexandre J C, Kobarg Jörg
Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Campinas, SP, Brazil.
Biochem Biophys Res Commun. 2006 Jul 28;346(2):517-25. doi: 10.1016/j.bbrc.2006.05.152.
Protein arginine methylation is an irreversible post-translational protein modification catalyzed by a family of at least nine different enzymes entitled PRMTs (protein arginine methyl transferases). Although PRMT1 is responsible for 85% of the protein methylation in human cells, its substrate spectrum has not yet been fully characterized nor are the functional consequences of methylation for the protein substrates well understood. Therefore, we set out to employ the yeast two-hybrid system in order to identify new substrate proteins for human PRMT1. We were able to identify nine different PRMT1 interacting proteins involved in different aspects of RNA metabolism, five of which had been previously described either as substrates for PRMT1 or as functionally associated with PRMT1. Among the four new identified possible protein substrates was hnRNPQ3 (NSAP1), a protein whose function has been implicated in diverse steps of mRNA maturation, including splicing, editing, and degradation. By in vitro methylation assays we were able to show that hnRNPQ3 is a substrate for PRMT1 and that its C-terminal RGG box domain is the sole target for methylation. By further studies with the inhibitor of methylation Adox we provide evidence that hnRNPQ1-3 are methylated in vivo. Finally, we demonstrate by immunofluorescence analysis of HeLa cells that the methylation of hnRNPQ is important for its nuclear localization, since Adox treatment causes its re-distribution from the nucleus to the cytoplasm.
蛋白质精氨酸甲基化是一种不可逆的翻译后蛋白质修饰，由一个至少包含九种不同酶的家族（称为PRMTs，即蛋白质精氨酸甲基转移酶）催化。尽管PRMT1负责人类细胞中85%的蛋白质甲基化，但其底物谱尚未完全明确，蛋白质底物甲基化的功能后果也尚未得到充分了解。因此，我们着手利用酵母双杂交系统来鉴定人类PRMT1的新底物蛋白。我们能够鉴定出九种参与RNA代谢不同方面的与PRMT1相互作用的蛋白，其中五种先前已被描述为PRMT1的底物或与PRMT1功能相关。在新鉴定出的四种可能的蛋白质底物中，有一种是hnRNPQ3（NSAP1），该蛋白的功能涉及mRNA成熟的多个步骤，包括剪接、编辑和降解。通过体外甲基化分析，我们能够证明hnRNPQ3是PRMT1的底物，并且其C端RGG盒结构域是甲基化的唯一靶点。通过使用甲基化抑制剂Adox进行进一步研究，我们提供了hnRNPQ1 - 3在体内被甲基化的证据。最后，我们通过对HeLa细胞的免疫荧光分析证明，hnRNPQ的甲基化对其核定位很重要，因为Adox处理会导致它从细胞核重新分布到细胞质中。
