The RAN GTPase

描述（由申请人提供）：RCC1是Ran GTPase的鸟嘌呤核苷酸交换因子。它在所有真核细胞中起着至关重要的作用-核运输，纺锤体形成，核膜形成和初级纤毛形成。它还与衰老、癌症和病毒感染有关。RCC1与染色质结合，在有丝分裂染色体周围产生RanGTP梯度。我们发现了一种新的RCC1的翻译后修饰，其中初始Met残基被切除，暴露的1-氨基被甲基化。单甲基化RCC1突变体引起有丝分裂缺陷。我们已经确定了负责这种修饰的1- n -甲基转移酶，我们将其命名为NRMT。NRMT甲基化的其他有趣靶点包括肿瘤抑制蛋白RB。NRMT的沉默导致有丝分裂缺陷。考虑到RCC1功能的关键重要性，1- n甲基化对其的调节，以及这种不寻常的修饰的高生物学意义，我们计划关注以下目标：当前的RCC1调节模型是否正确？已经提出RCC1在染色质上和染色质上动态循环，作为其催化作用的重要组成部分，并且n端尾部的磷酸化稳定了有丝分裂过程中染色质的结合。我们将严格测试这个模型，用栓系融合蛋白和磷酸化突变体取代内源性RCC1。我们将询问RCC1动力学在有丝分裂和细胞凋亡中是否重要。通过动物模型，我们还将测试Ran和RCC1是否参与肿瘤发生。2. 利用敲除小鼠和mef鉴定NRMT的生物学功能。我们将产生缺乏NRMT的KO小鼠，并询问小鼠是否显示染色体错误分离水平增加。为了确定RCC1与染色质关联减弱是否是主要缺陷，我们将在KO mef中表达染色质系住的RCC1，并询问我们是否挽救了正常的有丝分裂。我们提出假设，这种修饰的一般功能是促进染色质结合。RB的缺失会导致基因组的不稳定性，我们将测试一个不可甲基化的RB是否会导致类似的缺陷，以及这些缺陷是否与它向着丝粒募集CAP-3D有关。3. 1- n甲基化控制机制的确定。NRMT的核定位可能限制对某些靶蛋白的访问，从而阻止它们被甲基化。为了验证这个想法，我们将通过表达具有核输出信号的酶的一个版本来“敲开”NRMT。甲基化蛋白将通过质谱法与表达野生型NRMT的细胞进行比较。第二个假设是存在细胞质去甲基化酶。我们将使用细胞质提取物来纯化这种酶的活性。最后，我们发现在表达E7癌蛋白的HeLa细胞中，RB未被检测到甲基化。我们将探讨在这些细胞中阻断RB甲基化的潜在机制。

DESCRIPTION (provided by applicant): RCC1 is a guanine nucleotide exchange factor for the Ran GTPase. It plays vital roles in all eukaryotic cells - in nuclear transport, spindle formation, nuclear envelope formation, and primary cilium formation. It has also been implicated in aging, cancer, and viral infection. RCC1 associates with chromatin, and generates a RanGTP gradient around mitotic chromosomes. We identified a new post-translational modification of RCC1, in which the initiating Met residue is excised, and the exposed 1-amino group is methylated. Mon- methylatable mutants of RCC1 cause mitotic defects. We have identified the 1-N-methyltransferase responsible for this modification, which we named NRMT. Other interesting targets for methylation by NRMT include the tumor suppressor protein RB. Silencing of NRMT causes mitotic defects. Given the pivotal importance of RCC1 function, its regulation by 1-N-methylation, and the high biological significance of this unusual modification, we plan to focus on the following aims: 1. Are current models for RCC1 regulation correct? It has been proposed that RCC1 cycles dynamically on and off chromatin, as an essential part of its catalytic action, and that phosphorylation of the N-terminal tail stabilizes chromatin association during mitosis. We will rigorously test this model, by replacing endogenous RCC1 with tethered fusion proteins and phosphorylation mutants. We will ask if RCC1 dynamics are important in mitosis and apoptosis. Using an animal model, we will also test whether Ran and RCC1 are involved in tumorigenesis. 2. Identification of the biological functions of NRMT, using a knockout mouse and MEFs. We will generate KO mice lacking NRMT, and ask if the mice display increased levels of chromosome mis- segregation. To determine if weakened association of RCC1 with chromatin is the primary defect we will express a chromatin-tethered RCC1 in KO MEFs and ask if we rescue normal mitosis. We propose the hypothesis that a general function for this modification is to facilitate chromatin binding. Loss of RB causes genomic instability, and we will test if a non-methylatable RB causes similar defects, and if such defects are connected to its recruitment of CAP-3D to centromeres. 3. Determination of control mechanisms for 1-N-methylation. The nuclear localization of NRMT might limit access to certain target proteins, thereby preventing them from being methylated. To test this idea, we will "knock sideways" the NRMT by expressing a version of the enzyme that possesses a nuclear export signal. Methylated proteins will be compared by mass spectrometry to cells expressing wild type NRMT. A second hypothesis is that there exists a cytoplasmic demethylase. We will use cytoplasmic extracts to purify such an enzyme activity. Finally, we discovered that in HeLa cells, which express the E7 oncoprotein, RB is not detectably methylated. We will address the underlying mechanism that blocks RB methylation in these cells.