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

Abstract

The heterodimeric splicing factor U2AF plays an important role in 3 splice site selection, but the division of labor between the two subunits in vivo remains unclear. In vitro assays led to the proposal that the human large subunit recognizes 3 splice sites with extensive polypyrimidine tracts independently of the small subunit. We report in vivo analysis demonstrating that all five domains of spU2AF LG are essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology. A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in Schizosaccharomyces pombe. As this is not predicted by the model for metazoan 3 splice site recognition, we sought introns for which the spU2AF LG and spU2AF SM make distinct contributions by analyzing diverse splicing events in strains harboring mutations in each partner. Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3 pyrimidine tract. These and other studies performed in fission yeast support a model for 3 splice site recognition in which the two subunits of U2AF functionally collaborate in vivo. INTRODUCTIONThe removal of noncoding introns and the joining of coding exons via splicing, an essential step in the eukaryotic premRNA processing pathway, requires accurate splice site selection by the spliceosome. Initial recognition of the 5Ј exon/intron boundary is achieved via base pairing with the U1 snRNA component of the U1 snRNP (Zhuang and Weiner, 1986), whereas U2AF (U2 snRNP auxiliary factor) is the first factor bound to the 3Ј splice site (Ruskin et al., 1988). Biochemical complementation assays demonstrated that U2AF is required for the subsequent ATP-dependent association of U2 snRNP with pre-mRNA branchpoints (Ruskin et al., 1988;Valcarcel et al., 1996). 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/M...

Genomic mRNA Profiling Reveals Compensatory Mechanisms for the Requirement of the Essential Splicing Factor U2AF

“…For certain introns, such alternative interactions could provide the major contribution for early events during spliceosome assembly. A previous study on limited introns found no correlation between U2AF 59 requirement and the strength of the 5Ј splice site or other obvious features of pre-mRNAs; sequence features of the U2AF 59 -insensitive cdc16-I1 intron remained uncharacterized (58). Our study indicates the presence of a stronger 5Ј SS and a higher A/U percentage among the favorable features in U2AF 59 -insensitive introns ( Fig.…”

“…For this discussion of the U2AF 59 requirement(s) for splicing, we have classified cellular introns as U2AF 59 sensitive, U2AF 59 insensitive, and conditionally sensitive, as suggested by previous studies of specific introns (54,58). Similar to the behavior of well-studied model introns, the U2AF 59 -sensitive cdc2_b intron showed the conventional U2AF 59 requirement (Fig.…”

“…Relative to Saccharomyces cerevisiae, S. pombe shares many more features of pre-mRNA splicing with mammals: they both have degenerate splicing signals, their splicing factors (snRNAs and proteins) are similar to one another, and both require the two U2AF subunits (U2AF59 and U2AF23) (60,64). A temperature-sensitive allele of the S. pombe large U2AF subunit is lethal (42,46) and has been used to study splicing of specific transcripts in vivo (54,58). Thus, S. pombe offers an excellent model system to analyze the role of U2AF59 on a genomewide level.…”

A Conditional Role of U2AF in Splicing of Introns with Unconventional Polypyrimidine Tracts

“…In S. pombe, U2AF and BBP/SF1 form a stable complex (22). Unlike the mammalian introns, S. pombe introns show generally parallel requirements for both U2AF subunits (55). Moreover, various introns show FIG.…”

“…The heterozygous SpCR1 diploid (prp2::ura4/prp2 ϩ , ade6-M210/ade6-M216, ra4d18/ura4d18, leu1-32/leu1-32) and cdc2.2 and the Py tract variants (Y-long) (41), the prp2.1 mutant and the U2AF 59 genomic fragment plasmid pIRT3-prp2 ϩ (36), the U2AF 23 -ts mutant, (55), and the prp10-YH03 mutant (18) were previously described.…”