EXPRESSION AND FUNCTION OF U1 RNA ISOFORMS DURING DEVELOPMENT

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This proposal represents a continuation of our efforts to motivate promising minority students to seek careers in the biomedical sciences. To achieve this goal, students have been and will be trained in all aspects of molecular biology. The scientific project employed for this purpose involves the structural and functional characterization of U1- related sequences and their transcripts in the silkmoth Bombyx mori. U1 RNA is one of six prominent small nuclear RNAs (snRNAs), U1-U6, which are known to associate with six to ten proteins to form small ribonucleoprotein particles (snRNPs) which participate in the conversion of hnRNA to mRNA by splicing. Our laboratory has partially characterized three categories of U1-related genes in this organism. One category is composed of transcriptionally inactive pseudogenes; another, is represented by a highly reiterated U1-related sequences are developmentally and tissue-specific transcribed, suggesting a role in the regulation of gene expression. The experiments outlined in this proposal will allow our students to elucidate the role U1 isoforms play in the regulation of gene expression at the RNA splicing level. Their involvement in hnRNA splicing will be determined using developmentally staged silk glands, follicles, and several cell lines. U1 isoforms will be purified and used in vitro in reconstitution experiments in which their differential assembly into snRNP/hnRNP particles, association with intron-containing fibroin and chorion RNAs, the splicing capacities will be investigated. In vivo experiments will be performed to study the differential assembly and splicing efficiently of U1 isoforms in these staged cell types and cell lines. As with the case of beta-thalassemia RNA splicing mutations in humans, differential splicing as a result of U1 isoforms efficiency and/or specificity may prove to be the basis of some human maladies. The silkmoth , with a well-studies developmental and molecular biology, plus highly specialized cell types, provides a unique opportunity for our students to examine the function of these U1- related sequences. Specifically, minority students will be motivated to pursue a career in the biomedical sciences by exposing them to the pertinent literature, and training them to design experiments, formulate protocols, conduct experiments and publish the results.