The long term objective of this work is to understand the basic mechanisms involved in the control and catalysis of nuclear pre- mRNA splicing. This essential step in the expression of nearly all eukaryotic genes requires a precise removal of introns and joining of exons to generate functional messenger RNA. A number of genetic disorders are caused by aberrant splicing resulting from mutations in the splice site signals. Analogous mutations frequently cause defects in the splicing of mRNA precursors in vitro offering a convenient system to study factors determining splicing specificity at the molecular level. Splicing takes place in a multicomponent complex, the spliceosome, whose assembly involves formation and subsequent rearrangement of a number of intermediate complexes composed of small nuclear ribonucleoprotein particles (snRNPs) and splicing factors. A simplified in vitro system capable of specific recognition and precise splicing of RNA precursors will be used to study the mechanism of this reaction. In this system, the splice sites are provided in trans on two separate RNA molecules that can be extensively manipulated, facilitating the detailed analysis of their interactions with the spliceosome that determine the specificity of substrate recognition. Spliceosomal components present in the close proximity of the 5' splice site (5'SS) and thus, by definition, placed near the active site of the complex, will be detected by crosslinking analysis of the spliceosome using photoreactive derivatives of the 5155 RNA substrate. Selected crosslink products will be further characterized to identify and study the individual spliceosome components that interact with the 5'SS. In addition, a specific interaction between the p220 component of US snRNP and the invariant GU dinucleotide at the5' end of the intron will be analyzed to test if p220 is responsible for the remarkably specific recognition of the GU element during splicing. To study the mechanism of splicing catalysis, a battery of specifically modified 5'SS RNA substrates will be used. The modifications will be introduced at positions that are expected to interfere with the chemistry of splicing. Furthermore, a quantitative analysis of splicing reaction will be employed to build a kinetic framework of the system which is necessary for a precise interpretation of the results.
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