Nuclear precursor mRNA splicing takes place in a large, multicomponent complex, the spliceosome. spliceosome assembly involves a stepwise association of small nuclear RNP particles, snRNPs, and a number of other splicing factors, around the selected splice sites in pre-mRNA. Although the pre-mRNA sequence elements involved in splicing have been identified and well characterized, their role in controlling spliceosome assembly is not clear. We propose to study this problem using an in vitro system in which a specific association of snRNPs typical of an authentic spliceosome can be induced in the absence of pre-mRNA by the addition of the 5' splice site RNA consensus sequence. In the presence of the 5'SS RNA oligonucleotide, endogenous snRNP particles in the extract form a U2/U4/U5/U6 snRNP complex, the pseudospliceosome. This system provides a convenient way to study a partial reaction of the more complex spliceosome assembly process. We will study in detail the sequence requirements for this process as well as the nature of the target site for the interaction between the 5'SS RNA and snRNP components of the U2/U4/U5/U6 snRNP complex. While it is known that the 5' splice site sequence interacts by base pairing with the 5' end of U1 snRNA, this interaction is not required for the induction of the U2/U4/U5/U6 snRNP complex. Thus, another recognition site for the 5'SS sequence must exist. This site could reside within the U1 snRNP particle and/or the U2/U4/U5/U6 snRNP complex. We will test if U1 snRNP participates in the U2/U4/U5/U6 snRNP formation, and, if so, we will identify the elements within this particle (other than the 5' end of U1 snRNA) that are required for this process. We will also analyze and compare these two or more modes of 5' splice site recognition and assess their role(s) in mediating snRNP association and splicing. These findings allow us for the first time to establish a direct link between the recognition of the 5' splice site and the subsequent association of snRNPs into the spliceosome complex. Further studies should provide new insights into general mechanisms controlling snRNP associations and formation of splicing complexes.
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