The objective of this project is to characterize the functions of nucleolin, a nucleolar specific protein. Although nucleolin is thought to bind pre-ribosomal RNA, its actual function in processing nascent ribosomal RNA into 18S, 5.8S, and 28S ribosomal RNA remains unknown. Xenopus nucleolin consists of unique modules with distinct functional characteristics; the amino terminal third contains several alternating acidic and basic domains, while the carboxyl terminal two-thirds contains four RNA-binding domains. Xenopus oocytes will be injected with in vitro synthesized transcripts that encode either epitope-tagged full length Xenopus nucleolin or epitope-tagged nucleolin truncation peptides; subsequent immunolocalization of epitope-tagged nucleolin truncations to either the ribonucleoprotein matrices of lampbrush chromosome loops or subregions of multiple nucleoli will strongly indicate functional associations. Nucleolin's macromolecular associations within an established Xenopus pre-rRNA processing extract will be determined by immunoprecipitating nucleolin- containing ribonucleoprotein complexes from the extract. Associations with other nucleolar proteins or with nucleolar- specific small nuclear RNAs will expand our understanding of nucleolin function. Immunodepletion of nucleolin from the processing extract will also test the functional significance of nucleolin in the initial pre-RRNA processing reaction. These studies, which are at the interface of cellular and molecular biochemistry, will advance our fundamental knowledge of the function of the nucleus of the eukaryotic cell. %%% In eukaryotic cells, most of the genetic material is sequestered in the nucleus of the cell, and it is in the nucleus that the DNA is both replicated and transcribed into RNA, which in turn is transported out of the nucleus to function in the biosynthesis of proteins. Three different kinds of RNA function directly in protein biosynthesis in the cytoplasm: messenger RNAs, which contain the "codes" for the specific amino acid sequences of the nascent proteins; transfer RNAs, which are the "decoders" that supply the correct amino acids in the correct sequence; and ribosomal RNAs, which are structural components of the ribosomes, the machines which "sew" the amino acids together. While messenger RNA sequences are scattered throughout the DNA of the nucleus, the genes coding for ribosomal RNAs are grouped together in a microscopically-definable structural region of the nucleus called the nucleolus. The nucleolus contains, in addition to the ribosomal genes, a number of nucleolar-specific proteins which are presumed to be involved in either transcription or assembly of ribosomal RNAs, but for which specific functions have not yet been determined. In this project, the goal is to determine the function of one specific nucleolar protein, called nucleolin. This work will advance the understanding of a fundamental process of eukaryotic cells.
