The long term goals of the research program are to understand the mechanisms and control of eukaryotic gene expression with particular emphasis on the role of RNA-associated proteins in regulating post-transcriptional processing events. The current project, with Drosophila as the experimental system, uses two general experimental approaches to the problem: (a) molecular genetic analysis of the function of the major structural proteins (hnRNPs) associated with nuclear pre-mRNA, and (b) ultrastructural analysis of RNA transcription, ribonucleoprotein (RNP) formation, and early RNA processing events on nascent transcripts using the Miller chromatin spreading method. Results obtained over the last three years have laid the groundwork for a genetic test of function of this ubiquitous class of proteins, which are deposited on RNA as it is transcribed, forming an hnRNP complex that presumably serves as the substrate for subsequent processing events. Drosophila has two single copy genes encoding five similar basic hnRNP proteins of about 40 kDa, (and thus is quite similar to mammalian systems). One of these will be mutated so that an exogenous hnRNP gene can be introduced on a conditional promoter and the effects of hnRNP protein depletion studied. Sensitive genetic assays will allow study of subtle effects on transcription elongation, splicing and polyadenylation, all of which take place in intimate association with, or on, an hnRNP complex. Experiments are also planned to introduce altered copies of the gene so that the role of various protein isoforms (produced by alternative splicing), and various structural motifs in the protein (such as the RNP consensus sequence) can be analyzed in the living organisms. Immuno-EM with isoform-specific antibodies will indicate whether all forms of the proteins are on the transcripts of a given gene, or whether they bind in a gene-specific manner to RNA molecules. This approach should allow, for the first time, a direct test of the function of these ubiquitous and abundant proteins, whose role in pre mRNA metabolism is poorly-understood and somewhat controversial. Additional experiments are planned to continue EM ultrastructural analysis of the in vivo splicing process.
