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 spU2AFLG 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 spU2AFLG and spU2AFSM 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.

Figure 1.

Domain structure of spU2AF^LG and mutant phenotypes. (A) The relative lengths and positions of the conserved domains of spU2AF^LG are depicted as rectangles with amino acids or motifs targeted by mutations written above the wild-type schematic. Also included in the figure is the previously described ΔRS/Hinge (ΔL3-P189) mutant (Romfo et al., 1999). RS domain, diagonal stripes; linker region, black with the phenylalanine replacement of the key tryptophan (Kielkopf et al., 2001) shown in white; RRM1 and RRM2, cross-hatched with the RNP-1 motif shown on a white background with mutated amino acids in large type; ψRRM, stippled with the RNP1-like motif shown on a white background with mutated amino acids in large type. Phenotypes were assessed as described previously (Romfo et al., 1999; Webb and Wise, 2004). (B) Dominant negative effect on growth of overexpressing the ΔD2-P140 allele. A wild-type fission yeast strain (DS2) was transformed with the indicated plasmids in the presence of thiamine to repress the nmt1 promoter (Romfo et al., 1999). Growth was monitored by measuring absorbance₆₀₀ after a shift to medium lacking thiamine at time 0. In addition to the vector only and wild-type controls, we also analyzed growth of a strain harboring a plasmid carrying the ΔRS/Hinge allele, which we previously showed confers a recessive lethal phenotype (Romfo et al., 1999). (C) Two-hybrid analysis of heterodimer formation in mutants predicted to disrupt the large and small subunit interface. The figure shows the results of β-galactosidase assays, with the data expressed in Miller Units. Error bars, SD for n = 3. Wild-type, white bars; mutants, gray bars; negative controls, black bars.

Figure 2.

Microscopic analysis of the spU2AF^LG ΔP139-P189 and spU2AF^SM [C157S, C163S, H167I] mutants. (A) A haploid strain with a plasmid harboring wild-type spU2AF^LG covering a disruption of the uaf1+ locus (Romfo et al., 1999). (B) A haploid strain with a plasmid harboring the spU2AF^LG ΔP139-P189 mutant covering a disruption of the uaf1+ locus. (C) A haploid strain with a plasmid harboring wild-type spU2AF^SM covering a disruption of the uaf2+ locus (Webb and Wise, 2004). (D) A haploid strain with a plasmid harboring the spU2AF^SM [C157S, C163S, H167I] mutant covering a disruption of the uaf2+ locus. Magnification, ×1000.

Figure 3.

(A) Two-hybrid analysis of heterodimer formation in the [C157S, C163S, H167I] small subunit ZBDII mutant. Two-hybrid assays were performed as in Figure 1C. Wild-type, white bar; mutant, gray bar; negative control, black bar. (B) Plate assays to test the contribution to RNA binding of the [C157S, C163S, H167I] small subunit ZBDII mutant in the modified RNA three-hybrid system (Webb and Wise, 2004). The strength of the RNA-protein interaction is reflected by growth in the presence of increasing amounts of 3-amino-triazole (SenGupta et al., 1996; Eckman et al., 2002). The data shown are for the human β-globin 3′ splice site that is similar to many S. pombe 3′ splice sites; its sequence extending from just downstream of the branchpoint to 10 bases beyond the 3′ AG dinucleotide is shown beneath the plate assays.

Figure 4.

(A) Growth curve for the temperature-sensitive [Y108A, F111A] spU2AF^SM mutant. Growth of isogenic strains harboring either a wild-type or mutant allele was monitored by measuring the absorbance₆₀₀ of cultures propagated at the standard growth temperature (30°C) or at high temperature (37°C). Vertical blue arrows designate time points at which RNA was extracted. (B) RT-PCR assays of splicing in the ts spU2AF^SM mutant. (Top) Gel electrophoretic analysis of products from chromosomally expressed ypt5-I1 at three different time points after the shift to nonpermissive temperature and in parallel cultures propagated at the permissive temperature. The positions of linear pre-mRNA and mature mRNA are indicated schematically at the left. Bottom: histogram showing quantitation of RT-PCR data. White bars, percent precursor mRNA; black bars, percent mature mRNA. Error bars, SD for three RT-PCR splicing assays. Shown at the bottom is the sequence of the 3′ end of ypt5-I1 with the branchpoint sequence indicated in italics, the pyrimidine tract underlined, and the terminal AG in uppercase.

Figure 5.

RT-PCR assays of cdc16-I2, erf1-I2, and pyp3-I1 splicing in the ts spU2AF^SM and spU2AF^LG mutants. (A) Top left: gel electrophoretic analysis of products from cdc16-I2 in the spU2AF^SM [Y108A, F111A] mutant. Bottom left: histogram showing quantitation of RT-PCR data with percent precursor mRNA indicated by white bars and percent mature mRNA indicated by black bars. Error bars, SD for three RT-PCR splicing assays. (Right: as in the left panels except that the experiment was performed with the spU2AF^LG C387Y mutant. Bottom: the sequence of the 3′ end of cdc16-I2 with the branchpoint sequence indicated in italics, the pyrimidine tract underlined, and the terminal AG in uppercase. (B) As in A except that the splicing data are for erf1-I2. (C) As in A except that the splicing data are for pyp3-I1.

Figure 6.

Analysis of splicing in vivo for 22 introns in the ts spU2AF^SM and spU2AF^LG mutants. (A) Each number is followed by the gene name and sequence of the 5′ splice site, branchpoint, polypyrimidine tract, 3′ splice site, and three nucleotides beyond the AG at the intron/exon boundary. Complete sequences can be found at http://www/Sanger.ac.uk/Projects/S_pombe/. (B) Histograms showing quantitation of RT-PCR assays for the 22 introns depicted in A after heat inactivation of either subunit. Data were collected as in Figure 5 but are expressed as the percent precursor observed at 30°C subtracted from the percent precursor observed at 37°C (Δ precursor accumulation from 30 to 37°C). White bars, data for the spU2AF^SM [Y108A, F111A] mutant; black bars, the spU2AF^LG C387Y mutant data. Note that the scales are different for each subunit, owing to the more dramatic splicing defects caused by the large subunit ts allele at the 2 h time point (see text for details).