This proposal represents a continuation of our efforts to structurally and functionally characterize small nuclear RNAs (snRNAs) and spliceosomal proteins in the silk moth Bombyx mori. The research program's overall objective has been to provide information on the regulation of pre-mRNA splicing in an insect species. Our laboratory has structurally characterized variants of U1, U2 and U6 in the silk moth, and has started to study the proteins that differentially bind to them. The B. mori U1, U2 and U6 variants were found to be developmentally and tissue-specifically transcribed. In addition, we have demonstrated by UV cross-linking that specific nuclear proteins associate differentially with U1 variant sequences and certain U1 isoforms assemble into spliceosomal complexes preferentially. Based on the above results, two hypotheses can be entertained. One is that these snRNA variants are functionally significant and contribute to differential pre-mRNA splicing. The other postulates that the snRNA variants are functionally equivalent and that their tissue and developmentally specific expression results from unique interactions between their transcriptional controlling elements and cell-type specific trans-acting factors. The extreme and unparalleled polymorphism exhibited by B. mori in the form of multiple, tissue-specific, developmentally regulated U1, U2 and U6 variants, and the existence of tissues highly specialized for the production of a specific protein (fibroin or silk) make the silk moth an ideal model system to discriminate between the two hypothesis. This proposal describes experiments designed to investigate potential roles of U1, U2 and U6 variants as well as snRNP proteins in the control of gene expression during splicing. Their function in pre-mRNA splicing will be investigated using developmentally staged silk glands, follicles and several B. mori cell lines. Proteins differentially interacting with snRNA variants will be identified and sequenced. The 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. Due to the universal nature of the questions being asked, the answers obtained during the course of these experiments may have profound biomedical implications.
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