Immediately following the initiation of gene transcription by RNA Polymerase II, hnRNAs/pre-mRNAs are bound by a variety of nuclear RNA binding proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. Although these complexes are the substrates for all of the subsequent pre-mRNA processing steps which convert pre-mRNAs to mRNAs almost nothing is known about the role they play in mRNA biogenesis. This family of proteins is defined not only by unique protein primary sequence termed the ribonucleoprotein consensus sequence (RNP-CS), but also by the ability of some of its members to bind RNA in a sequence-specific fashion. It is the goal of this research to investigate the function of these nuclear RNA-binding proteins in Saccharomyces cerevisiae. This yeast provides ideal experimental tools to investigate the role of these proteins in nuclear RNA metabolism since it allows the complementary approaches of genetics and biochemistry to be used. This application proposes to isolate yeast nuclear hnRNA/pre-mRNA binding proteins using modifications of methods that have already been successfully developed for other eukaryotic cells. These proteins will then be used to prepare both polyclonal and monoclonal antibodies. The antibodies will be employed to isolate genomic clones, to learn about the primary structure of the proteins, and to study protein-protein and protein-RNA interactions important in the pre-mRNA processing pathway. The isolated genes will also be used for genetic analyses including gene disruption/deletion experiments to determine whether individual nuclear RNA binding proteins are required for cell growth. Genes will also be recloned under the control of inducible promoters and reinserted into yeast cells in which the wild-type copy has been deleted to examine the effect of regulated gene expression on nuclear RNA metabolism. Site-specific and domain-specific mutations will be constructed in vitro and conditional mutations isolated. As a longer term objective these mutant strains will b employed to screen for supressor mutations to determine what other types of nuclear factors interact with each protein. This approach may elucidate fundamental mechanisms used by eukaryotic cells to regulate gene expression at the post-transcriptional level.
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