Functional and Structural Roles of Nucleolin in Vertebrate Cell Nucleoli

在每一个真核细胞的细胞核中，都有一个形态上不同的区域，称为核仁，这在光学显微镜下很容易看到。核仁的主要功能是产生核糖体。核糖体是将遗传密码（以信使RNA的形式）翻译成特定蛋白质序列的机器，从化学上讲，它是由确定的蛋白质和核糖核酸分子（r-RNA）组成的超分子复合物。r-RNA分子的基因（DNA）位于核仁中并在核仁中转录，核糖体的某些组装过程也在核仁中进行。尽管我们知道核仁是制造核糖体的位置，但对于核仁特异性蛋白与小核仁RNA之间，或核仁特异性蛋白与核仁内基因转录的新生核糖体RNA之间发生的分子相互作用，我们所知相对较少。此外，人们对核仁蛋白在rna前加工、核糖体组装或核仁结构中的功能贡献知之甚少。核仁蛋白是一种丰富的具有模块化结构的核仁蛋白。已知核蛋白与核糖体前RNA结合，但对其功能或与其他细胞成分的相互作用知之甚少。这个项目的主要目标将是通过基因工程培育出一系列哺乳动物细胞（CHO细胞），其中核蛋白的合成可以通过在培养基中添加或减少药物来随意开启或关闭（例如，使用糖皮质激素诱导的MMTV启动子或四环素反激活剂系统）。由此产生的修饰细胞系将用于确定内源性核蛋白在体内的寿命，并表征缺乏内源性核蛋白的核仁结构。这些信息将作为后续研究（超出本项目范围）的基础，以确定在缺乏内源性野生型核蛋白的情况下表达改变版本的核蛋白的生物学后果。

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.