The long term objective of our research is to understand the way in which the nuclear pore controls all traffic entering the exiting the nucleus. In recent years, there has been an explosion in knowledge of the cascade of events which occur in the transduction of a signal from the plasma membrane to the nucleus. Increasingly, questions of signal transduction, developmental determination, oncogenic transformation, and HIV-1 viral infectivity are focusing on nuclear import or export. Unfortunately, we know relatively little of the molecular nature of the pore and how it controls this complex traffic. In the past grant period, we developed and used systems by which the nuclear pore and nuclear envelope can be reconstituted. Biochemically altered nuclear pores can now be created, following immunodepletion of the systems, and analyzed for function. Using such systems, we have identified a key intranuclear and pore glycoprotein, nup97, that is required for nuclear growth, DNA replication, and RNA export. We have also made good progress on delineating the assembly of the nuclear pore. Lastly, we have achieved as long-term goal, transcription from cloned templates assembled into synthetic nuclei, and ar now ready to examine RNA export in vitro. Our proposed goals focus on three areas: 1) A detailed analysis of the newly discovered nucleoporin, nup97. Specifically, we will clone the nus97 gene, create deleted and mutant nup97 proteins, and use the proteins to ask which domains are required to restore nuclear growth and DNA replication to 97-depleted nuclei. We will pursue in vivo studies to determine which domains of nup97 are involved in RNA export, and will identify the pore proteins and cytosolic factors that interact with nup97 through a powerful yeast genetic system. 2) A second major goal will be to molecularly dissect the steps in assembly of the nuclear pore using two systems, an annulate lamellae (AL) assembly system, and a novel anchored nuclear assembly system. These studies will help to elucidate the order of addition of pore proteins during assembly, the nearest-neighbor interactions, and the novel mechanism of membrane fusion which must occur across the double nuclear membrane to create a nuclear pore. Reconstituted annulate lamellae will also be used as a starting point for the first isolation of the vertebrate nuclear pore. Purification of the pore should allow production of monoclonal antibodies for identification of new pore proteins and provide previously unattainable insight into pore structure. 3) In the last section of the grant, we describe plans to achieve pol II and pol III transcription in synthetic nuclei, and to devise an assay for the in vitro RNA export of these transcripts. Once this is achieved, this system will allow us to analyze the mechanism of RNA export and the role of specific proteins, such as nup97, following immunodepletion and assembly of synthetic nuclei. In summary, we will pursue studies of pore function, pore assembly, and RNA export, with the goal of elucidating their role in cellular function.
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