The aim of the proposed research is to investigate certain aspects of the molecular mechanism of gene expression in mammalian and other eukaryotic cells. We propose to continue our studies of the organization of nuclear ribonucleoprotein complexes containing hnRNA (hnRNP) sequences, and the role these structures play in the correct processing and splicing of mRNA sequences. Our previous results suggest a structure for hnRNP in which the large RNA molecule is folded on distinct protein complexes to give a repeating 30S RNP substructure. Analysis by 2-D electrophoresis and immunoblotting using our monoclonal antibodies has now revealed that the major component of the 30S RNP substructures consist of a family of related polypeptides. We intend to determine the nature of these hnRNP core polypeptides by direct biochemical methods and by isolation of cDNA clones specific for them. The latter will also be used to study the structure and expression of the genes for these proteins. The hnRNP core protein pattern changes during the cell cycle, and the variations in the stoichiometry and metabolic properties suggest that they may be significant factors in the modulation of growth and differentiation of normal and transformed cells. We intend to determine the biochemical basis for these changing patterns of RNP proteins and attempt to determine their physiological significance using monoclonal antibodies and purified proteins in cell-free assays of RNA transcription and processing. To facilitate the assessment of the biological significance of our cell-free experiments, antibodies and purified proteins will be introduced into living somatic cells and oocytes. The immunocytochemical studies will be continued at the electron microscope level to more precisely locate RNP proteins within the cell and on spread chromosomes.
