In eukaryotic cells, messenger RNAs (mRNAs), the functional translatable intermediates of gene expression, are formed by extensive processing of primary gene transcripts (hnRNAs). We and others have recently demonstrated that hnRNAs exist in the nucleus in association with a specific set of proteins to form unique structures termed hnRNP particles. The hnRNP particles, one of the most abundant structures in the nucleus, are the sites of RNA processing. The RNP proteins are essential for the packaging, processing and function of these polynucleotides. The RNA-associated proteins can be photochemically crosslinked to it in intact cells, and the crosslinked complexes can be readily isolated. We have immunized mice with crosslinked hnRNP complexes and have generated monoclonal antibodies against several of the major hnRNP proteins in divergent eukaryotes. With these antibodies we have begun to characterize the hnRNP proteins and have isolated the hnRNP complex. One of the antibodies to the prominent hnRNP C proteins (41K and 43K) specifically inhibits the splicing of mRNA precursors in vitro. We shall investigate the structure and function of the hnRNP proteins and the hnRNP particles in order to understand their role in mRNA formation and in the packaging and organization of hnRNA in the nucleus. The antibodies will be used to isolate the cDNA clones for the hnRNP proteins by immunological screening of expression vector libraries. The amino acid sequence of the proteins will be determined form the cDNA nucleotide sequence and their mRNAs and their genes will be characterized. Additional antibodies to hnRNP proteins will be obtained and characterized. The interaction of hnRNP proteins with specific RNAs synthesized in vitro will be studied in order to understand the assembly and disassembly of the hnRNP complex -- knowledge which is necessary for understanding the structure and formation of hnRNP particles and the process of nucleo-cytoplasmic transport of mRNA. The function of the proteins and the hnRNP complexes in transcription and in RNA splicing will be studied in in vitro systems. The ability to isolate pure and intact hnRNP particles and the availability of antibodies to individual hnRNP proteins will enable the further characterization of their structure and their role in mRNA biogenesis.
