The long term goals of my research program are to understand the mechanisms and control of pre-mRNA processing events, with particular emphasis on the role played by RNA-associated proteins (hnRNPs and snRNPs). Using an unusual approach to this problem, the """"""""Miller"""""""" chromatin spreading technique for electron microscopy, we have visualized the process of spliceosome formation in vivo on specific nascent transcipts. It appears that splice site selection occurs very early and through the interaction on RNP components with splice junction signals. The research outlined herein will yield a manipulatable system for the study of the RNP template for RNA processing, using Drosophila as the experimental organism. We will first develop a system for the EM visualization of specific active genes. Two approaches will be investigated - (a) microinjection of genes on plasmid vectors into Drosophila embryos, and (b) germline transformation of Drosophila with genes linked to an amplification control element - both resulting in multicopy, ultrastructurally-distinguishable genes. The genes will be manipulated in vitro to yield duplications or changes in proximity of splicing signals. We will then analyze some of the requirements for spliceosome assembly in vivo, the kinetics of spliceosome formation on different introns, and the influence of the order of transcription and proximity of splice site sequences on splice junction pairing. Immuno EM analysis will locate specific snRNPs and hnRNP proteins on the RNP complex. Lastly, we will begin to clone the genes encoding Drosophila hnRNP proteins. This will allow (a) an evolutionary comparison to these proteins in higher eukaryotes, (b) a means to obtain antibodies to the Drosophila proteins for immuno EM studies, and (c) a genetic test for the function of the proteins.
