Moraes Karen C M, Quaresma Alexandre J C, Maehnss Karen, Kobarg Jörg
Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Rua Giuseppe Máximo Scolfaro 10.000, CP 6192, Campinas, SP, CEP 13084-971, Brazil.
Biol Chem. 2003 Jan;384(1):25-37. doi: 10.1515/BC.2003.004.
The mRNAs that encode certain cytokines and proto-oncogenes frequently contain a typical AU-rich motif that is located in their 3'-untranslated region. The protein AUF1 is the first factor identified that binds to AU-rich regions and mediates the fast degradation of the target mRNAs. AUF1 exists as four different isoforms (p37, p40, p42 and p45) that are generated by alternative splicing. The fact that AUF1 does not degrade mRNA itself had led to the suggestion that other AUF1 interacting proteins might be involved in the process of selective mRNA degradation. Here we used the yeast two-hybrid system in order to identify proteins that bind to AUF1. We detected AUF1 itself, as well as the ubiquitin-conjugating enzyme E2I and three RNA binding proteins: NSEP-1, NSAP-1 and IMP-2, as AUF1 interacting proteins. We confirmed all interactions in vitro and mapped the protein domains that are involved in the interaction with AUF1. Gel-shift assays with the recombinant purified proteins suggest that the interacting proteins and AUF1 can bind simultaneously to an AU-rich RNA oligonucleotide. Most interestingly, the AUF1 interacting protein NSEP-1 showed an endoribonuclease activity in vitro. These data suggest the possibility that the identified AUF1 interacting proteins might be involved in the regulation of mRNA stability mediated by AUF1.
编码某些细胞因子和原癌基因的信使核糖核酸（mRNAs）通常在其3'非翻译区含有一个典型的富含AU的基序。蛋白AUF1是首个被鉴定出能与富含AU区域结合并介导靶信使核糖核酸快速降解的因子。AUF1以四种不同的异构体（p37、p40、p42和p45）形式存在，这些异构体由可变剪接产生。AUF1自身不会降解信使核糖核酸这一事实表明，其他与AUF1相互作用的蛋白可能参与了选择性信使核糖核酸降解过程。在此，我们利用酵母双杂交系统来鉴定与AUF1结合的蛋白。我们检测到AUF1自身，以及泛素结合酶E2I和三种RNA结合蛋白：NSEP-1、NSAP-1和IMP-2，它们都是与AUF1相互作用的蛋白。我们在体外证实了所有相互作用，并绘制了参与与AUF1相互作用的蛋白结构域。用重组纯化蛋白进行的凝胶迁移实验表明，相互作用蛋白和AUF1能同时结合到富含AU的RNA寡核苷酸上。最有趣的是，与AUF1相互作用的蛋白NSEP-1在体外显示出核糖核酸内切酶活性。这些数据表明，所鉴定出的与AUF1相互作用的蛋白可能参与了由AUF1介导的信使核糖核酸稳定性调控。
