The major features of the normal axial and bipolar budding patterns (see Introduction
) are illustrated in and , lines 1 and 2. As reported by Ni and Snyder 
, we found that deletion of IST3
had no detectable effect on the axial budding pattern of haploid cells (data not shown) but profoundly affected the bipolar budding pattern of homozygous diploid mutants. In the mutants, use of the birth-scar-distal pole for the first bud on daughter cells was largely, although not entirely, lost (, lines 3 and 4), and cells that had budded multiple times showed many scars that were not at either pole; these scars were often present in chains reminiscent of those in axially budding cells ().
Budding-pattern phenotypes of wild-type and mutant strains.
Positions of first bud sites on daughter cells of wild-type and mutant strains.a
This phenotype resembled the inefficient axial budding seen in diploid cells when bipolar budding is disabled by mutation of RAX1, RAX2,
or both BUD8
; , line 5; ), suggesting that the ist3
mutants might be generally defective in generating the signals for bipolar budding. However, examination of the localizations of Bud8, Bud9, and Rax2 in the mutants revealed patterns indistinguishable from those seen in wild-type cells (). These observations suggested the alternative hypothesis that the ist3
mutants might have a partially functional axial-budding system in diploid cells, such as what occurs when Axl1 is ectopically expressed in such cells (see Introduction
; , line 6; ). In support of this possibility, ist3
mutant diploids showed normal bipolar budding when a gene important for axial budding was also deleted (, lines 7–9; ). Moreover, when the chromosomal AXL1
gene was tagged at its 3′ end with GFP
, the Axl1-GFP fusion protein was not detectable in wild-type diploid cells (as expected), but it was present in its normal (for haploid cells) localization at the mother-bud neck and division site in ist3
mutant diploid cells, although its levels appeared lower than those in wild-type haploid cells (, top four panels).
Expression and localization of cortical marker proteins in wild-type and mutant strains.
The accumulating evidence that Ist3 and Bud13 are involved in pre-mRNA splicing (see Introduction
) suggested that these proteins might be particularly important for splicing the pre-mRNA of MATa1
. This gene contains two introns 
and encodes one subunit of a heterodimeric repressor that normally prevents the expression of haploid-specific genes (such as AXL1
) in diploid cells 
. In this case, the expression of other haploid-specific genes should also be derepressed by a lack of Mata
1 in ist3
mutants. Indeed, a halo assay showed that the mating pheromone α-factor, which is normally expressed only in Matα haploid cells (, sectors 1–3), was also expressed in ist3
mutant diploid cells (, sectors 4 and 5). The levels of α-factor secreted by the mutants appeared to be somewhat lower than those from a wild-type Matα strain, suggesting that there might be some residual Mata
1 in the mutant cells.
α-factor production by bud13Δ and ist3Δ diploid strains.
To ask directly if Ist3 and Bud13 are involved in the splicing of MATa1
pre-mRNA, we used RT-PCR to examine the levels of the pre-mRNA and its splice products in various strains. In wild-type cells, most MATa1
RNA was present as the fully spliced product (, lane 10), although a significant amount of partially spliced product was also detected, reflecting differentially efficient splicing of the two introns, as reported previously 
. In contrast, although fully spliced product was detected in the ist3
mutant cells, its amount was greatly reduced relative to the partially spliced and unspliced RNAs (, lanes 11 and 12; note co-migration of the band derived from unspliced pre-mRNA with the band generated from contaminating DNA as PCR template when DNase treatment was omitted: , lanes 2–5). If the ist3
mutant budding-pattern phenotypes reflect a reduced level of Mata
1 protein due to inefficient splicing of MATa1
pre-mRNA, then expression of an intron-free MATa1
cDNA under control of a constitutive promoter should suppress the mutant phenotypes, and this was indeed the case (, lines 10 and 11; ).
Inefficient splicing of MATa1 pre-mRNA, but not of other pre-mRNAs, in bud13Δ and ist3Δ mutants.
To ask if the effects of ist3 and bud13 mutations are specific to MATa1 pre-mRNA, we also examined the splicing of the ACT1, RPS17A, DYN2, and RPL7A pre-mRNAs; the latter two genes were chosen specifically because, like MATa1, they contain two introns. Using primers derived from exon sequences and thus capable of amplifying both spliced and unspliced mRNAs (, open arrowheads), we saw little or no effect of the mutations on pre-mRNA processing for any of these four genes (, lanes 2–5 and 7–10; , lanes 2–6 and 8–12), in striking contrast to MATa1. For ACT1 and RPS17A, we also examined the products obtained using forward primers derived from intron sequences (such that only the unspliced pre-mRNAs could be amplified: , closed arrowheads). In each case, we saw a modest but significant increase in the amount of pre-mRNA-derived product (, lanes 2–6 and 8–12). Taken together, these data suggest that Ist3 and Bud13 play a major role in the splicing of MATa1 pre-mRNA but only a minor role in the splicing of many other pre-mRNAs that contain either one or two introns.
If Ist3 and Bud13 played a major role in the splicing of pre-mRNAs other than those we tested, it would be likely that at least one of the affected genes would be important for vegetative growth, so that the ist3 and bud13 mutants would show a significant growth defect relative to wild type. However, we observed only modest effects of the mutations on growth rates on either solid or liquid medium at 24°C or 30°C (). In contrast, a much larger effect was seen at 37°C (); although other explanations for this difference are possible, one plausible interpretation is that Ist3 and Bud13 may play a more important general role in pre-mRNA splicing at higher growth temperatures.
Growth rates of wild-type and mutant strains.
Strong evidence now suggests that Ist3 and Bud13 function, at least in part, in a retention-and-splicing (RES) complex with a third protein, Pml1 
. However, we could observe no effect of a pml1
mutation on bipolar budding (, line 12; ), expression of Axl1p or α-factor (, bottom panel; , sector 6), the splicing of any of the five pre-mRNAs tested (, lane 13; , lanes 6 and 11; , lanes 7 and 13; , lanes 7 and 13), or growth rate at any of the three temperatures tested (). These data suggest that at least in our strain background, Pml1 either does not play a major role or is functionally redundant for its role in the function of Ist3 and Bud13 in the splicing of either MATa1
or other pre-mRNAs.