The long term goal is to study and characterize the pre-mRNA splicing enzymes from human cells. Splicing is a critical step in maturation of mRNA precursors and most likely plays a major role in regulation of gene expression. The importance of studies on pre-mRNA splicing and their relation to clinical problems is underscored by the fact that several forms of alpha- and beta thalassemia result from aberrant splicing of human globin pre-mRNA. To elucidate the details of the splicing reaction and to characterize the splicing factors involved in this process the following specific aims are proposed: (1) To determine the effects of the structure of the pre-mRNA substrate on the efficiency of the splicing reaction. In particular the effects on the efficiency of splicing of the sequences surrounding the introns will be studied. 2. To study the in vitro splicing reaction in homologous systems. The hypothesis will be tested that in vitro splicing is more efficient in homologous systems or in extracts prepared from cells which express abnormally high levels of mRNA. 3. To purify and identify selected splicing factors. This study will focus on the activities involved in cleavage at the 5' splice site and on the splicing factor which can rescue a low salt inactivated splicing extract. Since sn- and hnRNPs appear to be involved in splicing, partially purified fractions will be tested for presence of sn- and hnRNPs to determine their specific functions in the splicing reaction.
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