RIBONUCLEOPROTEINS, MRNA AND CYTOSKELETAL STRUCTURES

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Messenger RNAs (mRNAs), the intermediates of gene expression which direct protein synthesis, are found in eukaryotic cells associated with a specific subset of cytoplasmic proteins. The mRNA-protein complexes, termed mRNP particles (mRNPs), are distinct structural entitles different in protein composition, structure, function and subcellular compartmentalization from nuclear pre-mRNA-protein (hnRNP) particles. A plethora of recent observations indicate that mRNP proteins play a cardinal role in mRNA transport, translation, stability and localization. The mRNP proteins can be photochemically crosslinked to the mRNA in intact cells and the crosslinked complexes can be readily isolated. We have studied these complexes in normal and virus-infected cells across eukaryotes. In all of these, the most abundant protein is a 72,000 dalton protein which is crosslinked to the poly(A) tail of the mRNA. The poly(A) binding protein is a highly conserved, poorly immunogenic protein that has been the focus of much interest because it appears to play a key role in mRNA metabolism. We have immunized mice with crosslinked purified mRNP complexes to produce antibodies to the mRNP proteins. Antibodies were obtained to the poly(A) binding protein from yeast, and we have begun to characterize the protein and have isolated the gene encoding it. The antibodies and the gene are the first probes for this protein from any organism and they open the way of its molecular and genetic characterization. We shall investigate the structure, function, properties and localization of the mRNP proteins and the mRNP complexes with particular emphasis on the poly(A) binding protein. Additional antibodies to mRNP proteins will be produced. The cDNAs encoding these proteins will be isolated by immunological screening of expression vector libraries. The complete amino acid sequence of the proteins will be determined by nucleotide sequencing of the cDNAs, and their mRNAs and genes will be characterized. The interaction of the proteins with RNA will be studied in detail. The subcellular localization of the proteins will be examined by immunocytochemical techniques. The functions of the proteins will be investigated in vivo by gene disruption and mutagenesis in yeast and by introduction of antibodies into living animal cells, and in vitro using cell free systems for protein synthesis and mRNA transcription, splicing and polyadenylation. From these studies a better picture of ho mRNA is formed, maintained and functions in animal cells is likely to emerge.