The experiments outlined in this renewed application continue the central theme of this project over the past 11 years. Since the start, our project has involved the structural and functional characterization of U1 and U2 genes, their transcripts and spliceosomal proteins in the silkmoth, Bombyx mori. The program's primary research objective has been to elucidate pre-mRNA splicing in an insect species. Over the years, this scientific program has served particularly well and will continue to be an excellent vehicle to educate and motivate promising minority students to pursue careers in the biomedical sciences. U1 and U2 RNAs are two of six abundant U-series small nuclear RNAs (snRNAs), U1-U6, which are known to associate with six to ten proteins. These RNA/protein complexes form small ribonucleoprotein particles (U-snRNPs) which function in splicing hnRNA (pre-mRNA) into mRNA /protein complexes form small ribonucleoprotein particles (U-snRNPs) which function in splicing hnRNA (pre-mRNA) into mRNA. Our laboratory has structurally characterized variants of U1 and U2 in the silkmoth, and started to examine the proteins that differentially bind to them. The U1 and U2 variants are developmentally and tissue-specifically transcribed, suggesting a role in control of gene expression. This proposal describes experiments designed to elucidate the role U1, U2 and snRNP protein variants play in the control of gene expression. Their function in pre- mRNA splicing will be investigated using developmentally staged silk glands, follicles and several B. mori cell lines. UV cross-linking experiments will be used to detect proteins with differential interactions to different U1 and U2 and snRNA variants. Following the UV cross- linking experiments, immunoprecipitation will be performed to identify and isolate the proteins in question. In addition, U1 and U2 isoforms characterized in our laboratory will be used in reconstitution experiments in which their differential interaction with spliceosomal proteins assembly into snRNP/hnRNP particles, association with in vitro transcribed intron- containing fibroin and chorion RNAs, and splicing capabilities will be ascertained. These studies should help define the suspected role of developmental and tissue-specific expression of U1 and U2 variants in differential splicing and development. The extreme and unparalleled polymorphism exhibited by B. mori in the form of multiple, tissue- specific, developmentally regulated U1 and U2 variants, and the existence of tissues highly specialized for the production of a specific protein (fibroin or silk) make the silkmoth an ideal model system. Due to the universal and central nature of the questions being asked, the answers obtained during the course of these experiments may have profound biomedical implications.
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