A conditional role of U2AF in splicing of introns with unconventional polypyrimidine tracts.

In animals, removal of introns from the majority (>80 to 90%) of nascent transcripts via pre-mRNA splicing is an important step for gene regulation (19). Alternative splicing serves important biological roles in diverse developmental contexts and provides an important mechanism to generate molecular diversity (7). The 5′ splice site (SS), the branch point sequence (BPS), and the polypyrimidine (Py) tract 3′ SS in the pre-mRNA are important splicing signals; they are recognized by the U1 snRNP, the U2 snRNP, and the U2 snRNP auxiliary factor U2AF, respectively, resulting in the formation of a dynamic RNA-protein complex called the spliceosome (23). In humans, the essential splicing factor U2AF is a heterodimer of a large protein subunit (U2AF65) and a small protein subunit (U2AF35). U2AF65 binds to the Py tract (62), and U2AF35 recognizes the 3′ SS (33, 61, 65). Both subunits of U2AF are essential for viability in many model organisms, such as the zebrafish, the fruit fly, the nematode worm, and fission yeast (U2AF59) (14, 24, 36, 43, 56, 66). However, in budding yeast the large subunit is dispensable (1) and the small subunit is absent. U2AF65 interacts with other splicing factors such as BBP/SF1, UAP56, SAP155 (or SF3b155), and SRp54 (1, 10, 15, 39, 64). The branch point binding protein BBP/SF1 binds to the BPS and cooperates with U2AF65 for RNA binding (2, 6). Detailed in vitro biochemical analyses using model splicing substrates in metazoans have significantly contributed to our mechanistic view of the role of U2AF65 function in splicing. The N terminus of U2AF65 harbors an arginine-serine-rich (RS) activation domain, and its C terminus contains three RNA recognition motifs (RRMs), each with a four-stranded antiparallel β-sheet and two α-helices (62). In metazoans, binding to the Py tract serves as the primary determinant for U2AF recruitment onto pre-mRNA. This interaction positions the RS domain to engage in a series of interactions with pre-RNA during spliceosome assembly, including stabilization of the base pairing between the BPS and the U2 snRNA (46, 53). U2AF65 is also an important target for splicing regulation, where splicing regulators such as SXL, PTB, hnRNP A1, ASF/SF2, SC35, and TRA can facilitate or antagonize its activity (7, 48). Whereas U2AF65 is highly conserved from fission yeast to humans, its C-terminal RRM3 domain is the only recognizable portion in the budding yeast protein Mud2p (1). The human RRM3 interacts with BBP/SF1 and SAP155 (or Schizosaccharomyces pombe prp10) (1, 15), and RRM3-related domains are present in several splicing factors (27). Deletion of the conserved RRM3 domain of the large subunit of U2AF (U2AF59) is lethal in S. pombe (4). Intriguingly, RRM3 shows no detectable RNA binding and is not required for the splicing of model substrates in a HeLa cell nuclear extract. RRM1 and RRM2 domains of the human U2AF65 are sufficient for Py tract recognition and in vitro splicing (4). Previously, we proposed that the RRM3 domain might be important for the splicing of only a subset of introns in vivo. Relative to Saccharomyces cerevisiae, S. pombe shares with mammals many more features of pre-mRNA splicing, including the presence of degenerate splicing signals, similarity of splicing factors (snRNAs and proteins), and a requirement for both subunits of U2AF (U2AF59 and U2AF23) (57, 59). Thus, by combining the power of genomics, molecular genetics, and biochemical analysis, S. pombe represents an excellent model system for analysis of the role of U2AF and Py tract in vivo. Our study of splicing in S. pombe offers important new information on RRM3 and U2AF functions and Py tract requirements in vivo. There is a large diversity in the arrangement of intronic sequences relevant for 3′ splice site recognition, and in the requirements for U2AF subunits and domains, beyond what is known from detailed in vitro biochemical analysis of model pre-mRNAs in metazoans. These findings also help explain why deletion of RRM3 is lethal in S. pombe whereas deletion of the human RRM3 has no effect on RNA binding and on the splicing of model substrates in vitro. Finally, whereas splicing of introns that lack a Py tract remains dependent on U2AF59, upstream Py tracts, located between the 5′ splice site and the BPS, are required for splicing in vivo only under conditions of U2AF59 inactivation.