The long term goal of this project is to study and to characterize the mRNA splicing enzymes from human cells. Splicing is a critical step in maturation of mRNA and most likely plays a major role in regulation of gene expression. The importance of this process is underscored by the fact that several forms of alpha- and beta- thalassemia result from aberrant splicing of human beta-globin mRNA. In order to study the splicing enzymes an efficient in vitro system for carrying out the splicing reaction is required. The immediate aim of this project is to develop such an efficient system. Initially whole cell extracts from HeLa cells will be used as a source of the splicing enzyme; human beta-globin RNA transcribed in vitro in the same extract will be used as a substrate. The spliced products will be analyzed by the primer extension assay. Two types of factors will be manipulated in order to improve the efficiency of the in vitro splicing system: the splicing machinery per se and the structure of the substrate. To this end the following specific aims are proposed: (1) To optimize the reaction conditions for the splicing enzymes by varying the concentration of ions, cofactors and protein components. (2) To elucidate the role of small nuclear ribonucleoproteins (snRNPs) in splicing, by studying the effects of purified snRNPs and antibodies directed against them on the in vitro splicing reaction. (3) To study the effect of the structure of the substrate (the number and position of introns, the poly A tracts and sequences located downstream from poly-adenylation sites) on in vitro splicing. (4) To fractionate the whole cell HeLa extract in order to isolate and characterize the splicing enzymes. (5) The results of the above research will be applied to study the splicing enzymes in the human leukemic cell line-K562 and in human methotrexate resistant cells. The substrates studied will be human gamma-globin mRNA and dihydrofolate reductase (DHFR) mRNA, respectively. Since genes for gamma-globin and DHFR are expressed in these cell lines, these two systems will constitute a model for splicing in a homologous system as opposed to the heterologous system used above (HeLa cells and beta-globin RNA).
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