CONTROL OF CELL FUNCTION VIA SELECTIVE MRNA TRANSLATION

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A complete understanding of the molecular mechanisms of disease will require elucidation of control mechanisms which define cell structure and function. The goals of this project are to define the molecular basis of selective messenger RNA translation and to characterize features of mRNA sequence and/or structure which signal formation of a specific messenger RNA protein complex. Stressed or virus-infected cells selectively translate a subset of the total mRNA pool, and nucleotides in the 5' untranslated leader sequence of the messenger RNAs facilitate differential expression. A plant viral messenger RNA, alfalfa mosaic virus RNA 4, will be exploited for these studies because it is expressed efficiently and selectively in extracts prepared from human cells infected with poliovirus wherein translocation of other messenger RNAs is abolished. Identification of the sequence and/or structural features of alfalfa mosaic virus RNA 4(AMV RNA 4) which sustain translational activity in diseased cells will increase understanding of how messenger RNAs are normally selected for translation and may suggest protocols for combating viral infection or for prevention of cell shut-down during stress conditions. Preliminary studies show that signals which permit selective translation. A combination of nucleotide deletion experiments and peptide complementation analyses will be used to characterize the molecular basis of selective AMV RNA 4 translation. Despite the implication that specific messenger RNA- protein interactions form the basis of important regulatory mechanisms, very few examples of specific mRNA-protein complexes have been described an studied in detail. Preliminary studies demonstrate that two unrelated proteins bind specifically to a 3'- terminal fragment of AMV RNA 4. Analysis of the specific binding of the AMV coat protein (CP) and of the mammalian double strand RNA-dependent protein kinase (dsI) to a 180 nucleotide fragment of RNA 4 offers an uncommon opportunity to examine mRNA protein interactions. Characterization of the AMV RNA 4-CP complex will be useful for understanding the molecular basis of viral cross- protection in plants; moreover, preliminary studies show that the plant viral RNA fragment is a potent activator of the mammalian dsI kinase. Mobility band shift electrophoresis and nitrocellulose filter retention studies will be used to characterize the specific interaction between alfalfa mosaic virus RNA 4 and CP/dsI. Nucleotide deletion analysis and site-directed mutagenesis will be used to probe both the RNA and the protein binding sites. The availability of large quantities of CP permits extension of the analysis to include physical studies. Direct clinical relevance may be found in recent reports detailing significant changes in the development of a neoplastic phenotypic associated with features of the 5' untranslated leader sequence of oncogene mRNAs; moreover, interactions of protein(s) with the 5' leader of the human immunodeficiency virus mRNAs influence expression of viral gene products.