Alternative pre-mRNA splicing is an important mechanism for regulating gene expression during normal human development. Derangements in the splicing process underlie some genetic diseases. In the red cell, key mechanical and morphological properties of the membrane skeleton are influenced by alternative splicing choices that occur in progenitor cells. In particular, developmentally regulated alternative splicing of protein 4.1R exon 16 (E16) governs synthesis of isoforms with either low affinity (in early progenitors) or high affinity (in late progenitors) for spectrin and actin. The applicants long-term objectives are to understand the molecular """"""""splicing switch"""""""" that activates inclusion of exon 16 during erythropoiesis, allowing synthesis of high affinity isoforms of 4.1R, and to apply this knowledge to a larger understanding of alternative splicing in erythroid cells. In model 4.1R pre-mRNA constructs spliced in vitro and in cells, previous studies show that the process of exon 16 splicing occurs in a specific sequential order, beginning with excision of the downstream intron. This process is under positive and negative control by specific splicing enhancer and silencer elements in 4.1R pre-mRNA. These elements are a modular and include: a splicing enhancer in exon 16, an adjacent silencer in exon 16, a putative splicing enhancer in intron 16 that may activate tissue-specific downstream intron splicing, and a unique """"""""derepressor"""""""" sequence in exon 17 that is essential for upstream intron splicing. To understand how these multiple components work coordinately and dynamically to regulate the ordered splicing of exon 16, the following specific aims are proposed: (1) to define more precisely the size and sequence-specificity of each RNA regulatory element; (2) to identify the RNA splicing factor proteins that bind to each site, explore functional interactions among these proteins, and assess their impact on selected early steps of spliceosome assembly, and (3) to test the general relevance of the 4.1 splicing model to erythroid alternative splicing by reconstituting 4.1 splicing in mouse erythroleukemia cells and by identifying other erythroid-specific splicing events. Experimental strategies will include mutagenesis and domain-swapping experiments, extensive use of RNA-protein interaction techniques, purification of potentially novel splicing factors, and bioinformatics approaches to sequence analysis.
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