The RAN GTPase

描述(由申请人提供)：这项资助的持续目标是在分子水平上了解RAN GTP酶和RAN相互作用蛋白在核运输和有丝分裂中的功能。除了它们基本的生物学相关性外，这些蛋白质在病毒感染、衰老和癌症中发挥着关键作用。特别是，我们最近在RAN交换因子RCC1方面的工作表明，它对于维持基因组稳定是必不可少的。在之前的资助期间，我们确定了Exportin-5和Importin-11的功能，并深入了解了全球对核运输的监管。我们还发现了一种新的、完全没有预料到的RCC1翻译后修饰类型，它调节与染色质的关联。鉴于这一新修饰的关键重要性，我们计划在下一个资金阶段专注于这一领域，通过以下4个目标：1.确定调节RCC1功能的机制。我们发现RCC1在其N端的a-氨基上发生甲基化。我们还发现，核提取液含有以前未被认识的a-N甲基转移酶活性，并且RCC1的甲基化增强了染色质的结合。主要目的是确定这种不寻常的修饰是否对RCC1有额外的影响；确定a-N-甲基转移酶(a-N-MT)；并确定其活性是否受到调控。2.RCC1的构象转换。RCC1与染色质的结合受RAN的调节。为了了解a-N-甲基化的潜在机制和作用，我们将使用FRET生物传感器测试RCC1的N-末端在RAN结合时是否发生构象变化。3.鉴定RCC1在有丝分裂中的Ran非依赖性功能。突变的RCC1，D182a，在随机结合、核苷酸交换活性和染色质结合方面存在缺陷，但在细胞中表达时仍会诱导有丝分裂缺陷。这些数据表明，RCC1具有独立于其全球环境基金活动的功能。我们将测试N端尾巴是否是诱导有丝分裂缺陷所必需的或足够的，确定尾巴中的关键残基，并寻找介导RCC1这些影响的结合伙伴。4.确定未甲基化的RCC1是否导致基因组不稳定。原代小鼠胚胎成纤维细胞将产生稳定的细胞系，以表达野生型RCC1或甲基化缺陷突变体。这些细胞系将进行核型分析，以鉴定非整倍体，并将在体外测试遗传不稳定性，以及在小鼠体内形成肿瘤。RCC1突变体将提供一种严格的新方法来检验肿瘤发生的非整倍体假说。

DESCRIPTION (provided by applicant): The continuing goal of this grant is to understand at a molecular level the functions of the Ran GTPase and of Ran-interacting proteins in nuclear transport and mitosis. In addition to their fundamental biological relevance, these proteins play key roles in viral infection, aging, and cancer. In particular, our recent work on the Ran exchange factor, RCC1, suggests that it is essential for maintaining genomic stability. In the previous funding period we determined the functions of Exportin-5 and Importin-11, and gained insight into the global regulation of nuclear transport. We also discovered a new, entirely unanticipated type of post-translational modification of RCC1 that regulates association with chromatin. Given the pivotal importance of this novel modification, we plan to focus on this area during the next funding period, through the following 4 aims: 1. Identify the mechanisms that regulate RCC1 function. We have discovered that RCC1 is methylated on its N-terminal a-amino group. We also found that nuclear extracts contain a previously unrecognized a-Nmethyltransferase activity, and that the methylation of RCC1 enhances chromatin association. Key goals are to detemine whether this unusual modification has additional effects on RCC1; to identify the a-Nmethyltransferase (a-N-MT); and to determine whether its activity is regulated. 2. Conformational switching by RCC1. RCC1 binding to chromatin is modulated by Ran. To understand the underlying mechanisms and the role of a-N-methylation, we will test whether the N-terminus of RCC1 undergoes a conformational change upon Ran binding, using FRET biosensors. 3. Identify Ran-independent functions of RCC1 in mitosis. A mutant RCC1, D182A, is defective in Ranbinding, nucleotide exchange activity, and chromatin binding, but nonetheless still induces mitotic defects when expressed in cells. These data argue that RCC1 possesses a function that is independent of its GEF activity. We will test whether the N-terminal tail is necessary or sufficient to induce mitotic defects, identify critical residues within the tail, and seek binding partners that mediate these effects of RCC1. 4. Determine whether unmethylated RCC1 induces genomic instability. Stable cell lines will be generated from primary murine embryonic fibroblasts, to express either wild-type RCC1 or a methylation-defective mutant. These cell lines will be karyotyped to identify aneuploidies, and will be tested for genetic instability in vitro, and for tumor formation in mice. The RCC1 mutants will provide a rigorous new way to test the aneuploidy hypothesis of tumorigenesis.