PILOT STUDY--NUCLEOLAR CYTOPLASMIC TRANSPORT

点击翻译按钮获取中文摘要

Exchange of macromolecules between the nucleolus and the cytoplasm is an essential process of all eukaryotic cells. We previously identified Nopp140, a nuclear localization signal binding protein that shuttles between the nucleolus and the cytoplasm on highly localized tracks. The long-term objective of this grant proposal is to understand the mechanism and regulation of Nopp140 mediated nucleolar-cytoplasmic transport. Specifically: 1. Characterization of p57 and identification of additional Nopp140 associated proteins. We have identified a Nopp140 associated protein (p57) in rat liver nuclear extracts that localized to Nopp 140 like intranuclear tracks, and cloned its cDNA. We propose to: (a) Determine if p57 also shuttles between nucleus and cytoplasm. (b) Study the interaction of endogenous and bacterially expressed p57 with Nopp140. (c) Identify additional Nopp 140 interacting proteins by affinity chromatography of cellular fractions using columns of recombinant Nopp140 and p57. 2. Structure and dynamics of Nopp 140 tracks. To develop a system that allows an easier detection of the tracks and that will enable us to simultaneously manipulate cellular incubation conditions, we will try to visualize them on a light microscopical level by laser scanning confocal microscopy and three dimensional reconstruction of optical sections of: (a) immunolocalized Nopp140 and p57, and (b) fluorescently labeled recombinant Nopp 140 and p57, after microinjection or in in vitro nuclear import assays. 3. Molecular Analysis of the Nopp140 gene. Nopp140 is encoded by two mRNAs that are differentially expressed in different cell types but transcribed from a single gene. To gain insight into the expression pattern of the two mRNAs, the Nopp140 gene will be cloned from a rat genomic lamba DASH library and its promoter and intron-exon structure analyzed. Ultimately, these studies will contribute to the understanding of the upregulation of ribosome synthesis and the resulting nucleolar hyperactivity which are major characteristics of every cancer cell. In fact, the importance of the nucleolus in the regulation of cell growth has been emphasized by the recent discovery of the nucleolar oncoprotein LYAR.