The Splicing Factor U2AF Small Subunit Is Functionally Conserved between Fission Yeast and Humans

Abstract

The small subunit of U2AF, which functions in 3 splice site recognition, is more highly conserved than its heterodimeric partner yet is less thoroughly investigated. Remarkably, we find that the small subunit of Schizosaccharomyces pombe U2AF (U2AF SM ) can be replaced in vivo by its human counterpart, demonstrating that the conservation extends to function. Precursor mRNAs accumulate in S. pombe following U2AF SM depletion in a time frame consistent with a role in splicing. A comprehensive mutational analysis reveals that all three conserved domains are required for viability. Notably, however, a tryptophan in the pseudo-RNA recognition motif implicated in a key contact with the large subunit by crystallographic data is dispensable whereas amino acids implicated in RNA recognition are critical. Mutagenesis of the two zinc-binding domains demonstrates that they are neither equivalent nor redundant. Finally, two-and three-hybrid analyses indicate that mutations with effects on large-subunit interactions are rare whereas virtually all alleles tested diminished RNA binding by the heterodimer. In addition to demonstrating extraordinary conservation of U2AF smallsubunit function, these results provide new insights into the roles of individual domains and residues.The heterodimeric splicing factor U2AF (U2 snRNP auxiliary factor) recognizes the 3Ј splice site in the earliest biochemically defined complex formed on premessenger RNAs (reviewed in reference 34). Human U2AF, initially identified as an activity necessary for the recruitment of U2 snRNP to the branchpoint (41), consists of a 65-kDa subunit (58) and a 35-kDa subunit (60). The large subunit of U2AF contacts the polypyrimidine tract, while the small subunit recognizes the 3Ј AG (27,55,58,64). Orthologs of both subunits are found in Drosophila melanogaster (21, 37), Caenorhabditis elegans (62, 63), Schizosaccharomyces pombe (33, 50), and Arabidopsis thaliana (8). While a poorly conserved ortholog of the U2AF large subunit, Mud2p, has been identified in Saccharomyces cerevisiae, the genome does not contain an open reading frame that resembles the small subunit (1). The absence of a U2AF small-subunit orthologue in S. cerevisiae versus its strong conservation in S. pombe (62% identity and 78% similarity with human U2AF35 over the first three domains; see Fig. 3) provides an impetus to understand better the function of this enigmatic splicing factor.Because human U2AF 35 is present in a stable complex with U2AF 65 , it was quite surprising when early studies provided evidence that the small subunit is dispensable for splicing in vitro (57). Subsequently, however, reconstitution experiments demonstrated that the small subunit is required for only a subset of splicing events in HeLa nuclear extracts whereas the large subunit appeared to be critical for excision of all introns tested (see, e.g., references 13, 15, 27, 61, 65). The small subunit of U2AF may also be dispensable for some splicing events in vivo, as none of the pre-mRNAs tested showed splicing defects in...

Analysis of Mutant Phenotypes and Splicing Defects Demonstrates Functional Collaboration between the Large and Small Subunits of the Essential Splicing Factor U2AF In Vivo

“…An even more notable phenotype of the spU2AF LG ⌬P139-P189 mutant is that individual cells harboring this deletion allele as their sole source of U2AF large subunit display an aberrant branched morphology (Figure 2, compare A and B). Strikingly, a triple substitution (C157S, C163S, H167I) within the second zinc-binding domain (ZBDII) of the S. pombe U2AF small subunit identified in a separate study (Webb and Wise, 2004) also exhibits slow growth and a branched morphology (Figure 2, compare C and D). Like the other 35 small subunit alleles analyzed to date (Webb and Wise, 2004), this mutant is recessive as judged by the reversal of its deleterious effects on introduction of a plasmid-borne wild-type allele.…”

“…Purified U2AF is a heterodimer composed of large and small subunits in humans (Zamore and Green, 1989), Drosophila melanogaster (Kanaar et al, 1993;Rudner et al, 1996), Caenorhabditis elegans (Zorio et al, 1997; Zorio and Blumenthal, 1999) and Schizosaccharomyces pombe (Potashkin et al, 1993;Wentz-Hunter and Potashkin, 1996). Functional conservation of this splicing factor is evidenced by 1) the restoration of splicing activity to U2AF-depleted HeLa nuclear splicing extracts via addition of Drosophila U2AF large subunit (dmU2AF LG ; Zamore and Green, 1991) and 2) the ability of human U2AF 35 (hsU2AF SM ) to restore growth to an S. pombe strain lacking the small subunit (Webb and Wise, 2004).The structural domains of U2AF are also conserved except in Saccharomyces cerevisiae, where the large subunit is highly divergent and the small subunit is absent entirely (Abovich et al, 1994). The small subunit of U2AF consists of two zinc-binding domains (ZBDs) surrounding a central pseudo-RNA recognition motif (⌿RRM; Rudner et al, 1998b), also known as a PUMP (PUF60/U2AF/MUD2 protein-protein interaction) domain (Page-McCaw et al, 1999) or a UHM (U2AF homology motif; Kielkopf et al, 2004), which are highly conserved between S. pombe and humans, followed by a C-terminal domain that consists of RS or RS/glycine repeats in metazoan orthologues, which are not present in the fission yeast protein.…”

“…After 2 h (2, 4, and 6 h for ypt5-I1) at the nonpermissive temperature, cells were harvested and total RNA extracted as previously described (Alvarez et al, 1996). Semiquantitative RT-PCR assays were performed and the results analyzed as in published work (Webb and Wise, 2004) using the primers (17-60) listed in Table 1. …”

“…Two-hybrid analysis was performed as previously described (Webb and Wise, 2004). Briefly, the fusion protein constructs were cotransformed into the S. cerevisiae strain PJ69 -4A 20) as described (James et al, 1996) except that dimethyl sulfoxide was added to the final incubation step. …”

“…The resulting fragments were inserted into pAS2-1 (BD Sciences Clontech, Palo Alto, CA) and sequenced. To express a fragment derived from the ␤-globin 3Ј intron/ exon boundary (Wu et al, 1999) in the modified three-hybrid system (Webb and Wise, 2004), the 5Ј phosphorylated oligonucleotides 15 and 16 were annealed and inserted into the XmaI and ClaI sites of p4130 (kindly provided by John Woolford, Carnegie Mellon University; described in Fewell and Woolford, 1999) to generate the plasmid ␤-globin/pIII MS2-1 B,H,C. …”