Spatial Organization of Protein-RNA Interactions in the Branch Site-3′ Splice Site Region during pre-mRNA Splicing in Yeast

The locations of introns within nascent yeast pre-mRNAs is specified largely by just three short conserved sequences located at and near the 5′ and 3′ ends of the intron (6, 47). These sequences are recognized during the assembly of the spliceosome, a massive macromolecular complex consisting of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) and at least 60 associated proteins (31, 57). The spliceosome not only must precisely recognize intron boundary sequences to avoid the introduction of catastrophic frameshift errors in coding regions but also must be able to accommodate significant variations in intron size, sequence, and organization. A full understanding of how accurate splice site recognition is achieved will require detailed knowledge of the dynamic contacts between the conserved sequences that define intron boundaries and the components of the spliceosome. Nuclear pre-mRNA splicing proceeds via two sequential transesterification reactions. In the first catalytic step, the 2′ hydroxyl group of the last adenosine residue in the conserved branch site sequence (UACUAAC) attacks the phosphodiester bond at the 5′ splice site to yield free 5′ exon and branched lariat intermediates. The branch site and 5′ splice site sequences are initially recognized by binding of the branch point bridging protein (BBP) (5) and the U1 snRNA (45), respectively. These sequences are then recognized a second time through interactions involving the U2 and U6 snRNAs prior to the first catalytic step (22, 23). In the second step, the 3′ hydroxyl of the 5′ exon intermediate attacks the phosphodiester bond at the 3′ splice site to produce ligated exons and release the excised intron lariat. Genomic analysis of intron sequences in the yeast Saccharomyces cerevisiae has shown that the conserved YAG↓ sequence that defines the 3′ splice site is located between 9 and 143 nucleotides (nt) downstream of the branch site sequence (47). The branch site-3′ splice site region is initially recognized through binding of the BBP and Mud2p, the yeast ortholog of the mammalian splicing factor U2AF65 (1, 5). Selection of the 3′ splice site for the second step is tightly coupled to the prior recognition of the branch site sequence, as the first AG downstream of the pre-mRNA branch site sequence is generally chosen, with an optimal spacing of 18 to 22 nt (10, 26, 37). Genetic analyses in yeasts have provided strong evidence that non-Watson-Crick interactions with the conserved ↓GUA at the 5′ splice site and the highly conserved GAG sequence in the U6 snRNA function in binding of the 3′ splice site YAG↓ for the second catalytic step (14, 36). Other genetic analyses have also suggested an important role for intron sequences adjacent to the 3′ splice site in 3′ splice site selection (37). Six yeast proteins, Prp8p, Prp16p, Prp17p, Slu7p, Prp18p, and Prp22p, have been shown to be involved in the second catalytic step (42, 53). Four of these proteins, Prp8p, Prp16p, Prp22p, and Slu7p, have each been reported to cross-link at or near the 3′ splice site (30, 48, 49, 52). U2 snRNA and ATP-dependent cross-linking of the 3′ splice site to an additional, unidentified ∼122-kDa protein has also been reported, but the role of this protein, if any, in 3′ splice site selection is unknown (30). The relevance of the cross-linking of Prp22p to the 3′ splice site during normal splicing is also unclear, as strong cross-linking of Prp22p has been reported to occur with mutant substrates unable to complete the second step (30). A central role for Prp8p has been suggested from the isolation of prp8 mutants that affect the fidelity of 3′ splice site recognition (13, 15, 50, 51). ATP hydrolysis by Prp16p, as part of a complex involving Prp17p, Slu7p, Prp18p, and Prp22p, facilitates a conformational rearrangement that involves the 3′ splice site and that leads to its protection from oligonucleotide-directed RNase H cleavage. This conformational rearrangement appears to occur in two stages, an initial ATP-dependent stage involving Prp16p and Prp17p and a subsequent ATP-independent stage involving Slu7p, Prp18p, and Prp22p (42, 53). Previous results suggested that the protection of the 3′ splice site from RNase H cleavage is most likely due to the Prp16-dependent interaction of Prp22p with the 3′ splice site (30). In a series of elegant experiments, Schwer and colleagues (5a, 43, 58) showed that the in vitro requirements for Slu7p, Prp18p, and Prp22p in the second step can be alleviated if actin-based pre-mRNA substrates containing short intervals between the branch site and the 3′ splice site are used. These results suggested that Slu7p, Prp18p, and Prp22p may act to form a bridge between or stabilize the interactions of factors bound to the branch site and the 3′ splice site (6, 43, 58; however, see reference 12). ATP hydrolysis by Prp16p has also been linked to the use of a discard pathway for aberrant lariat intermediates in which Prp16p acts as a clock to time the rate of a spontaneous conformational change linked to the identity of the branch site nucleotide (7, 8). To better understand the spatial organization and dynamics of protein-RNA interactions involved in branch site-3′ splice site selection in S. cerevisiae, we have developed a series of closely related, efficiently spliced cross-linking substrates. Using these substrates, we found that upon prespliceosome formation, the yeast ortholog of the mammalian U2-associated splicing factor SAP155 (19, 38) becomes associated with a large region encompassing the branch site, 3′ splice site, and 3′ exon sequences. Following the first catalytic step of splicing, the U5 snRNP-associated protein Prp8p associates extensively with the 3′ splice site region in a Prp16p-independent manner. Despite the previous implication of Prp16p in the recognition of the branch site nucleotide (7, 16), cross-linking of Prp16p to sequences within or near the branch site is not observed. Instead, cross-linking of Prp16p is restricted to the last four intron nucleotides together with at least 13 nt of the adjacent 3′ exon. Inhibition of the second catalytic step using 3′ splice site mutant substrates results in the accumulation of cross-links to Prp22p in the last eight intron nucleotides. Identical strong cross-linking of Prp22p to wild-type 3′ splice sites can be observed when the second step is blocked by phosphorothioate substitution of the nonbridging Rp oxygen at the 3′ splice site. These results demonstrate that the association of Prp22p with the 3′ end of the intron represents an authentic transient intermediate that normally exists prior to the second catalytic step. Finally, we provide evidence that an unidentified ∼80-kDa cross-linked species interacts specifically with intron sequences downstream of the branch site following exon ligation. The distinct spatial and temporal associations of these and other proteins with the branch site-3′ splice site region provide a detailed portrayal of the overall organization of this region during the initial stages of intron recognition, spliceosome assembly, and catalysis.