This revised proposal stems from work begun during a FIRST Award to the Applicant. The Investigator will further characterize the cis- and trans-acting factors that are involved in directing splicing of beta-spectrin pre-mRNA along erythroid- specific or non-erythroid pathways. The Investigator has identified the splice acceptor and donor sites for both pathways of pre-mRNA processing. He has established a minigene/transfection assay system that allows him to examine both tissue and developmental specificity of RNA processing. His plan of attack calls for deletional analysis as well as site-directed mutagenesis to approach the problem. He will use the information gained from examination of the cis-acting elements in the splicing process to construct RNA that contains the minimum required amount of sequence to direct splicing correctly. This construct will be used as a probe in the search for trans-acting factors in the system. The methodology will involve gel retardation studies with nuclear extracts from non-erythroid cells and erythroid cells at various stages of maturation. Other studies will involve UV cross-linking of the RNA probe to the transacting factors. The trans-acting factors will be isolated and identified. Previously undescribed factors will then be cloned.
