An understanding of the mechanisms and controls of eukaryotic gene expression is of critical importance to the larger goal of regulating these processess in diseased cell states. The controls that are operative on pre-messenger RNA at the post-transcriptional nuclear stage are being studied. During this period, pre-mRNA transcripts are packaged with hnRNP and snRNP components into a ribonucleoprotein complex which becomes the substrate for splicing and polyadenylation reactions; they are then exported from the nucleus as mature mRNAs for translation in the cytoplasm. The A/B hnRNP proteins are associated with these transcripts throughout the entire process--they bind to the RNA co-transcriptionally and they remain with it until after it transits the nuclear pore. There is much suggestive evidence that the A/B hnRNP proteins may play important roles in RNA packaging, processing and export, although definitive in vivo evidence has proven difficult to obtain. A very favorable system has been established in Drosophila melanogaster which will allow a combined genetic/cytological/ultrastructural approach to investigation of the function of these proteins in a living organism. Using recently obtained flies that have null mutations in both of the major A/B hnRNP protein genes, (and which are late embryonic lethals when homozygous), assays will be done of RNA transcription, processing and mRNA export as the protein levels fall below that required for normal function. These assays will be ultrastructural (Miller chromatin spreading), cytological and phenotypic. Flies will be established that have a conditionally expressed hnRNA protein gene so that these assays can be done in other tissues and developmental stages of interest. Guided by a wealth of available biochemical data on these proteins, genes expressing specific mutant proteins will be introduced into wild flies. In this background, they will be assayed for possible transdominant effects on RNA processing and export and for their specific patterns of binding to sites of transcription on polytene chromosomes. They will then be crossed into the double mutant flies and assayed for their ability to rescue viability. This combined approach will be a powerful in vivo test of the role of specific protein domains in RNA recognition, processing and export and will provide a critical test of the function of A/B hnRNP proteins in a living organism.
