The long term objectives of this research proposal are to understand the mechanisms involved in the processing and modification of pre-messenger RNA. Experiments are proposed to address three aspects of RNA processing: 1. The regulation of alternative splicing. 2. The recognition and pairing of splice sites in constitutively spliced pre- mRNA. 3. The editing of pre-mRNA by site-specific adenosine deamination. Pre-mRNA splicing is a critical step in the pathway of normal gene expression. Defects in splicing are the cause of numerous human genetic diseases and they are correlated with certain types of human cancer. The editing of pre-mRNA results in the production of proteins with amino acid sequences distinct from these encoded in genomic DNA. Defects in this process would have a profound effect on the function of the encoded proteins. The problem of regulated alternative splicing will be approached by a detailed functional and structural analysis of a well characterized splicing enhancer complex from the Drosophila doublesex pre-mRNA AN essential component of this complex is a member of the serine/arginine(SR) family of splicing factors which are highly conserved from flies to man. These proteins are thought to mediate interactions between the 5' and 3' splice site in constitutively spliced RNA and the interaction between splicing enhancer complexes and the basic splicing apparatus in alternatively spliced RNAs. A novel hybrid protein approach will be used to determine the function of the arginine-serine-rich region of SR proteins in alternative and constitutive splicing. This approach involves the use of low molecular weight reversible protein dimerizers that can be used to tightly control activity of hybrid SR protein during the splicing reaction. The role of exon sequences in splice site recognition will be approached by determining whether novel enhancer sequences recognized by specific members of the SR family of proteins correspond to conserved regions in the exons of beta-globin pre-mRNAs. In addition, UV crosslinking and mutagenesis studies will be carried out to identify and characterize the function of SR protein binding sites in the beta-globin exons The mechanism of pre-mRNA editing will be approached by studying interactions between a specific editing enzyme and two highly structured editing sites in GluR-B pre-mRNA. In addition, a novel immunoaffinity selection method will be employed to identify and characterize new targets for editing by site-specific adenosine deamination. When completed, the proposed studies will significantly advance the understanding of the mechanisms of RNA splicing and editing.
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