As part of a long term commitment to understand the structure and function of the mammalian erythrocyte, this laboratory is studying the genetic mechanisms that regulate synthesis of the unique constellation of membrane skeletal proteins expressed in red cells. One of those skeletal components, protein 4.1, is a multi-functional structural protein that interacts with several other skeletal elements and exhibits a broad spectrum of molecular weights in erythroid and nonerythroid cells. Multiple isoforms of this structural protein are translated from a family of differentially-spliced mRNAs that are derived from a single large 4.1 gene but have distinct tissue-specific expression patterns. This proposal seeks to investigate the role and mechanisms of alternative RNA splicing events responsible for tissue-specific expression of protein 4.1 """"""""spliceoforms."""""""" Erythroid-specific splicing events in two regions of the gene will be investigated: one regulates utilization of alternative translation initiation sites located in two different 5' exons of the gene, while a second controls expression of several structurally different spectrin-actin binding domains. Cellular models that reproduce developmental splicing switches in protein 4.1, such as mouse erythroleukemia (MEL) cells, will be developed; in vitro nuclear splicing extracts will be prepared by standard techniques; and splicing substrates in the form of pre-mRNAs transcribed from protein 4.1 genomic """"""""minigenes"""""""" will be synthesized. Experimental manipulation of these critical components will allow characterization of regulatory nucleotide sequences within the pre-mRNA, as well as putative trans-acting nuclear splicing factors that interact with these sequences. Ultimately, putative splicing factor cDNAs will be cloned using either specific probes (antibodies or oligonucleotides) or eukaryotic expression cloning techniques. Specific methodologies employed previously in characterization and cloning of DNA binding proteins, such as gel shift mobility assay, DNA-affinity chromatography, prokaryotic expression cloning with oligonucleotide probes, and eukaryotic expression cloning using biological activity assays, will be adapted for use in studying putative RNA binding proteins that mediate alternative splicing. Erythropoiesis constitutes a remarkable differentiation process in which nucleated erythroid precursor cells are extensively remodeled until they achieve the unique morphology characteristic of mature erythrocytes. The research proposed here should elucidate the mechanism(s) whereby alternative splicing effects important structural changes in red cell membrane proteins during erythropoiesis. In a broader sense, however, this proposal represents an exploration of a major question in hematology research, namely, what genetic strategies are utilized to effect the substantial changes in gene expression that underly the dramatic phenotypic differentiation in erythropoiesis? these studies will ultimately reveal whether alternative RNA splicing is a major effector of erythropoiesis, as in the Drosophila somatic sexual differentiation pathway, or simply an economical genetic mechanisms for changing the structure of selected membrane proteins.
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