In vitro assembly of an early spliceosome defining both splice sites

For splicingof a metazoan pre-mRNA, the four major splicesignals - 5′ and 3′ splicesites (SS), branch-pointsite (BS), and a poly-pyrimidine tract (PPT) - are initially bound by splicing factorsU1 snRNP, U2AF35, SF1, and U2AF65, respectively, leading up to an early spliceosomalcomplex, the E-complex. The E-complex consists of additional components and the mechanism of its assembly is unclear. Hence, how splicesignals are organized within E-complex defining the exon- intronboundaries remains elusive. Here we present in vitro stepwise reconstitution of an early spliceosome, assembled by cooperative actions of U1 snRNP, SRSF1, SF1, U2AF65, U2AF35, and hnRNP A1, termed here the recognition (R) complex, within which both splicesites are recognized. The R-complex assembly indicates that the SRSF1:pre-mRNA complex initially defines a substrate for U1 snRNP, engaging exons at both ends of an intron. Subsequent 5′SS-dependent U1 snRNPbinding enables recognition of the remaining splicesignals, defining the intron. This R-complex assembly indicates the minimal constituents for introndefinition revealing mechanistic principles behind the splicesite recognition.