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.

Public Health Relevance

The preservation of genetic stability during cell division is vital to life, and defects in stability can result in cancer and premature ageing. Cell division depends upon the formation of a gradient of RanGTP around the chromosomes, which in turn depends on the association of a guanine nucleotide exchange factor for Ran, called RCC1. We have discovered a novel enzyme that modifies RCC1 and helps retain it on chromosomes. Loss of this enzyme can cause mitotic defects and genetic instability. This enzyme also modifies an important tumor suppressor. Therefore, understanding the biological functions of this enzyme, and of RCC1, are important for understanding how cells protect themselves from premature ageing and cancer.

National Institute of Health (NIH)
Research Project (R01)
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Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
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Ainsztein, Alexandra M
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Vanderbilt University Medical Center
Anatomy/Cell Biology
Schools of Medicine
United States
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Rodriguez-Boulan, Enrique; Macara, Ian G (2014) Organization and execution of the epithelial polarity programme. Nat Rev Mol Cell Biol 15:225-42
Tooley, Christine E Schaner; Petkowski, Janusz J; Muratore-Schroeder, Tara L et al. (2010) NRMT is an alpha-N-methyltransferase that methylates RCC1 and retinoblastoma protein. Nature 466:1125-8
Eisinger-Mathason, T S Karin; Andrade, Josefa; Groehler, Angela L et al. (2008) Codependent functions of RSK2 and the apoptosis-promoting factor TIA-1 in stress granule assembly and cell survival. Mol Cell 31:722-36
Hao, Yi; Macara, Ian G (2008) Regulation of chromatin binding by a conformational switch in the tail of the Ran exchange factor RCC1. J Cell Biol 182:827-36
Dorfman, Julia; Macara, Ian G (2008) STRADalpha regulates LKB1 localization by blocking access to importin-alpha, and by association with Crm1 and exportin-7. Mol Biol Cell 19:1614-26
Goldstein, Bob; Macara, Ian G (2007) The PAR proteins: fundamental players in animal cell polarization. Dev Cell 13:609-22
Kremer, Brandon E; Adang, Laura A; Macara, Ian G (2007) Septins regulate actin organization and cell-cycle arrest through nuclear accumulation of NCK mediated by SOCS7. Cell 130:837-50
Riddick, Gregory; Macara, Ian G (2005) A systems analysis of importin-{alpha}-{beta} mediated nuclear protein import. J Cell Biol 168:1027-38
Plafker, Scott M; Plafker, Kendra S; Weissman, Allan M et al. (2004) Ubiquitin charging of human class III ubiquitin-conjugating enzymes triggers their nuclear import. J Cell Biol 167:649-59
Chen, Ting; Brownawell, Amy M; Macara, Ian G (2004) Nucleocytoplasmic shuttling of JAZ, a new cargo protein for exportin-5. Mol Cell Biol 24:6608-19

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