Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3′ splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein–protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions.

Human splicing factor SF3a is a component of the mature U2 small nuclear ribonucleoprotein particle (snRNP) and its three subunits of 60, 66, and 120 kDa are essential for splicing in vitro and in vivo. The SF3a heterotrimer forms in the cytoplasm and enters the nucleus independently of the U2 snRNP. Here, we have analyzed domains required for in vitro interactions between the SF3a subunits. Our results indicate that the SF3a66-SF3a120 interaction is mediated by a 27-amino acid region in SF3a120 C-terminal to the second suppressor-ofwhite-apricot and prp21/spp91 domain and amino acids 108 -210 of SF3a66. Neither of these sequences contains known structural motifs, suggesting that the interaction domains are novel. Moreover, an ϳ100-amino acid region, including the SURP2 domain of SF3a120 but extending into neighboring regions, is sufficient for binding to SF3a60. Analysis of determinants for nuclear import of SF3a demonstrates that SF3a120 provides the major nuclear localization signal and SF3a60 contributes to nuclear import.The U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) 5 play essential roles in pre-mRNA splicing by interacting with conserved sequences at exon-intron boundaries and assembling the catalytic core of the spliceosome (1). They consist of one or two uridine-rich small nuclear RNAs (snRNAs), seven common (Sm) and several particle-specific proteins (2). Whereas U6 snRNP biogenesis is entirely nuclear, newly transcribed U1, U2, U4, and U5 snRNAs are exported to the cytoplasm. Binding of the Sm proteins generates "core snRNPs," which are imported into the nucleus. The final maturation steps (snRNA modification and binding of particle-specific proteins) occur in Cajal bodies, followed by movement of the mature snRNPs to sites of splicing and storage (3, 4).Except for the U1 snRNP, other snRNPs exist in different forms. U4, U5, and U6 snRNPs undergo association-dissociation cycles that are important for spliceosome assembly (2). In addition, different forms of the U2 snRNP have been isolated. The human 12 S U2 snRNP consists of the U2 snRNA, the Sm proteins, and the U2-specific proteins U2-AЈ and U2-BЉ, whereas a 17 S U2 snRNP contains additional polypeptides representing subunits of splicing factors SF3a and SF3b (2, 5). In vitro, the 17S U2 snRNP assembles in a stepwise fashion (6). SF3b associates with the 12 S U2 snRNP to yield a 15 S intermediate, which is converted into the 17 S U2 snRNP upon SF3a binding. Only the 17 S U2 snRNP, but not the 12 S or 15 S particles, is functional (6, 7). SF3a and SF3b interact with the pre-mRNA at or in the vicinity of the branch site early during spliceosome assembly, which is thought to recruit and tether the U2 snRNP to the spliceosome (8 -10). At later stages of the splicing reaction, SF3a and SF3b appear to be destabilized from the U2 snRNP because they are underrepresented in the catalytically active spliceosome (11).SF3a comprises three evolutionarily conserved subunits (SF3a60, SF3a66, and SF3a120), all of which are necessary fo...