The RAN GTPase

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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.