Functional and Structural Roles of Nucleolin in Vertebrate Cell Nucleoli

点击翻译按钮获取中文摘要

9727917 DiMario In the nucleus of every eukaryotic cell, there is a morphologically distinct region termed the nucleolus, which can be seen readily by light microscopy. The primary function of the nucleolus is the production of ribosomes. The ribosome is the machine which translates the genetic code (in the form of messenger RNA) into specific protein sequences, and chemically it is a supramolecular complex consisting of defined proteins and ribonucleic acid molecules (r-RNAs). The genes (DNA) for the r-RNA molecules are located in the nucleoli and are transcribed there, and certain aspects of ribosomal assembly take place there. Although we know that nucleoli are the site in which ribosomes are made, relatively little is known about the molecular interactions that occur either between nucleolar-specific proteins and the small nucleolar RNAs, or between nucleolar-specific proteins and the nascent ribosomal RNA that is transcribed from genes within the nucleolus. Also, very little is known about the functional contributions made by nucleolar proteins in pre-RNA processing, ribosome assembly, or nucleolar structure. This proposal focuses on nucleolin, an abundant nucleolar protein with a modular structure. Nucleolin is known to bind to pre-ribosomal RNA, but little else is known about its function or its interactions with other cellular components. The primary objective of this project will be to genetically engineer a line of cultured mammalian cells (CHO cells) in which the synthesis of nucleolin can be turned on or off at will by the addition or subtraction of drugs from the culture medium (e.g., using either the glucocorticoid-inducible MMTV promoter or the tetracycline transactivator system). The resulting modified cell lines will be used to determine the life span of endogenous nucleolin in vivo and to characterize nucleolar structure in the absence of endogenous nucleolin. This information will be used as the basis for subsequent studies (beyond the scope of this project) to determine the biological consequences of expressing altered versions of nucleolin in the absence of endogenous wild-type nucleolin.