This proposal is a continuation of our work to structurally and functionally characterize the small nuclear RNAs (snRNAs) and their transcriptional controlling sequences in the silk moth Bombyx mori. The experiments outlined in this proposal are designed to elucidate the role that snRNA variants play in the regulation of gene expression at the pre-mRNA splicing level. Their involvement in pre-mRNA splicing will be assessed employing staged silk glands and follicles. Over the years, we have observed that: (1) the silk moth is endowed with populations of U1, U2, U4 and U6 snRNA isoforms; (2) these variants are expressed differentially in tissues and developmental stages; (3) specific single-stranded RNA binding proteins differentially bind to some snRNA isoforms; (4) certain variants preferentially associate with high molecular weight spliceosomal complexes. Taken together, these data suggest the following hypothesis: snRNAs are functionally significant and contribute to differential pre-mRNA splicing, tissue differentiation and development. Alternatively, these isoforms may be functionally equivalent. To test these two hypotheses, we propose the following specific aims: (1) to assess the relative abundance of snRNA isoforms in different tissues, stages of development and spliceosomal complexes; (2) to identify and characterize proteins that bind to snRNA variants; (3) to ascertain the in vitro assembly and splicing efficiency of snRNA variants with nuclear extracts from different tissues and developmental stages; (4) to analyze the assembly and splicing efficiency of snRNA variants in vivo. The extreme and unparalleled polymorphism exhibited by B. mori in the form of multiple, tissue-specific, developmentally regulated U1, U2, U4 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 hypotheses. Due to the universal nature of the questions being asked, the answers obtained during the course of these experiments may have profound biomedical implications given the number of human maladies in which alternative pre-mRNA splicing is highly significant. Thus, the expected differential functions of these variants may modulate development and tissue differentiation in diverse organisms including humans. In addition, the PI has formulated a detailed professional development plan designed to improve his competitiveness in procuring mainstream funding.