Characterization of a U2AF-Independent Commitment Complex (E′) in the Mammalian Spliceosome Assembly Pathway

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Abstract
Early recognition of pre-mRNA during spliceosome assembly in mammals proceeds through the association of U1 small nuclear ribonucleoprotein particle (snRNP) with the 5′ splice site as well as the interactions of the branch binding protein SF1 with the branch region and the U2 snRNP auxiliary factor U2AF with the polypyrimidine tract and 3′ splice site. These factors, along with members of the SR protein family, direct the ATP-independent formation of the early (E) complex that commits the pre-mRNA to splicing. We report here the observation in U2AF-depleted HeLa nuclear extract of a distinct, ATP-independent complex designated E′ which can be chased into E complex and itself commits a pre-mRNA to the splicing pathway. The E′ complex is characterized by a U1 snRNA-5′ splice site base pairing, which follows the actual commitment step, an interaction of SF1 with the branch region, and a close association of the 5′ splice site with the branch region. These results demonstrate that both commitment to splicing and the early proximity of conserved sequences within pre-mRNA substrates can occur in a minimal complex lacking U2AF, which may function as a precursor to E complex in spliceosome assembly.