The removal of introns from pre-messenger RNA (pre-mRNA) is a requirement for the synthesis of functional messenger RNA (mRNA) from many eukaryotic genes. Failure of the pre-mRNA splicing process can prevent gene expression and lead to disastrous consequences for the organism. Several human disorders, including a number of thalassaemias, result from splicing defects. In addition, pre-mRNA splicing is a regulated process which controls the normal expression patterns of numerous genes. The splicing reaction takes place on a large ribonucleoprotein (RNP) particle called the spliceosome. Within the spliceosome are a number of small nuclear RNAs (snRNAs) which, together with associated proteins, comprise the small nuclear RNP (snRNP) subunits of the spliceosome. The long term objective of this study is to determine the contribution of snRNPs to the rate and fidelity of pre-mRNA splicing. Specific attention will be placed on defining the role of the U1 snRNP particle in 5' splice site utilization. U1 snRNA contains a sequence of its 5' end which base pairs with the 5' splice site sequence of pre-mRNA molecules. The U1-specific, 70 kd protein binds U1 snRNA adjacent to this pre-mRNA binding domain. An immediate goal of this work will be to determine if the 70 kd protein influences 5' splice site recognition, cleavage, or ligation. The gene encoding the 70 kd protein will be isolated from Saccharomyces cerevisiae (yeast) and in vivo and in vitro assays will be developed to monitor 70 kd protein function. Genetic and biochemical experiments will be performed to characterize the RNA binding properties of the 70 kd protein and to determine the contribution of the U1 snRNA/70 kd protein complex to spliceosome formation and splicing.
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