Cellular function and regulation of RCC1 isoforms

Living organisms are made of cells. The genes that control all the functions of each cell are made of DNA and are collected in several chromosomes that are contained in the cell nucleus. Communication between the nucleus and the rest of the cell (the cytoplasm) is important for the production of proteins encoded by the genes. The interaction between the nucleus and the rest of the cell may be important for the spatial organisation of the cell, particularly during cell division, when the nucleus is dramatically reorganised. In animal and plant cells, the nuclear envelope, a membrane structure that surrounds the chromosomes, breaks down during cell division, and pairs of chromosomes become arranged on a structure called the spindle. One of each pair of chromosomes is separated to each end of the spindle, then this structure breaks down and the cell is pinched in two, leaving one set of chromosomes to form a new nucleus in each new cell. During cell division, the spatial orientation of the chromosomes and the spindle relative to the rest of the cell determines the plane of cell division. This is important in tissues, where cells are arranged relative to each other and their intracellular structures are polarised, that is arranged in a particular direction. A protein called Ran is important in the communication between the nucleus and the rest of the cell. The active form of Ran is made at chromosomes by another protein, RCC1. Active Ran directs the movement of other proteins and large molecules between the nucleus and cytoplasm. It also directs spindle assembly during mitosis and nuclear envelope formation. We have found that RCC1 exists in cells in at least three different forms (isoforms) that we think may have different functions and may be important when cells actively proliferate during development and in cancer. We will study how these different RCC1 isoforms work, using extracts of human cells and frog eggs which can reproduce complicated processes like spindle assembly and nuclear formation in a test tube. We will investigate the protein complexes made by RCC1 isoforms by a technique called mass spectrometry. We will also use advanced techniques for microscopy to study how RCC1 isoforms work in living cells that are grown on a Petri dish. To do this, we will make RCC1 isoforms in the cells coupled to a naturally fluorescent protein from jellyfish. This work will enable us understand how RCC1 isoforms work and how complicated processes in cells are coordinated by communication between the nucleus and cytoplasm. This work may help us to understand how to control cell division and tissue growth, and how they can go wrong in diseases.

Generation of Ran-GTP on chromsosomes plays critical roles in nucleocytoplasm transport, mitotic spindle formation and nuclear envelope assembly. Ran-GTP also determines the orientation of the spindle with the oocyte cortex during meiosis, and may therefore play additional roles in communication between the nucleus and the rest of the cell. Ran-GTP is generated by the highly specific guanine nucleotide exchange factor RCC1. We have identified at least three isoforms of RCC1 in human cells that differ in their N-terminal regions (NTRs) through alternative splicing of the mRNA. We have found that the alpha and gamma isoforms have remarkably different properties in their molecular interactions, regulation by phosphorylation and tissue expression. We propose that the different RCC1 isoforms have divergent functions that may control different cellular processes or regulate Ran-GTP production during cell proliferation or differentiation. This proposal seeks to determine the cellular functions of RCC1 isoforms. We will determine their cellular localisation, trafficking and roles in Ran-dependent processes. We will use Xenopus egg extracts as a near-physiological cell-free system to study spindle formation, nuclear envelope assembly and nuclear transport in vitro. We will use cultured human cells to determine the sub-cellular localisation, mobility and function of RCC1 isoforms during the cell cycle. We will examine the effects of the deletion and expression of specific isoforms in mammalian cells. We will determine the expression and localisation of RCC1 isoforms in normal and cancerous human tissues. Finally, we will examine the potential role of expression during cell proliferation and transformation. It is anticipated that this work will identify the cellular functions of specific RCC1 isoforms, and provide substantial advances in our understanding of the control of the Ran system, which plays a central role in cellular coordination.