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.
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