Mating Type Switching in Yeast Controlled by Asymmetric Localization of ASH1 mRNA

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Science  18 Jul 1997:
Vol. 277, Issue 5324, pp. 383-387
DOI: 10.1126/science.277.5324.383


Cell divisions that produce progeny differing in their patterns of gene expression are key to the development of multicellular organisms. In the budding yeast Saccharomyces cerevisiae, mother cells but not daughter cells can switch mating type because they selectively express the HO endonuclease gene. This asymmetry is due to the preferential accumulation of an unstable transcriptional repressor protein, Ash1p, in daughter cell nuclei. Here it is shown thatASH1 messenger RNA (mRNA) preferentially accumulates in daughter cells by a process that is dependent on actin and myosin. A cis-acting element in the 3′-untranslated region of ASH1mRNA is sufficient to localize a chimeric RNA to daughter cells. These results suggest that localization of mRNA may have been an early property of the eukaryotic lineage.

During early development, cellular diversity is achieved by differences between cells in their patterns of gene expression. A good example of differential gene expression in lower eukaryotes occurs during the diploidization of homothallic strains of the budding yeastSaccharomyces cerevisiae (1). Upon germination, haploid spores grow to a critical size and then produce buds. Anaphase takes place at the bud neck, and a complete set of chromosomes is delivered to both the mother cell and the daughter cell (bud). The mother cell can switch its mating type, but the daughter cell cannot. This difference is due to mother cell–specific transcription of theHO gene, which encodes an endonuclease that initiates gene conversion at the mating type locus (2).

Transcription of HO in mother cells is due to the unequal accumulation within daughter nuclei of a repressor of HOtranscription called Ash1p (3). This accumulation depends on at least five SHE genes, one of which (SHE1, also known as MYO4) encodes a type V myosin (3). Because mRNA localization is a common mechanism for generating asymmetric protein distribution in higher eukaryotes (4), we investigated the localization of ASH1 mRNA in the dividing yeast cell.

Using fluorescent in situ hybridization (FISH), we initially analyzed cells carrying the ASH1 gene on a multicopy plasmid (5, 6). ASH1 mRNA–specific fluorescence was detected in 7% of the cells from an asynchronous culture. In binucleate cells that had undergone anaphase but not cell separation,ASH1 mRNA was concentrated at the distal tips of buds. Depending on the genetic background, 35 to 66% of the binucleate cells with a FISH signal showed localization of ASH1 mRNA. WhenASH1 mRNA was not localized to the bud tip, it often appeared to form filamentous tracks extending from daughter cells into mother cells. In contrast to ASH1 mRNA, polyadenylated [poly(A)+] RNA was dispersed throughout both mother and daughter cells (Fig. 1A, top row). A null mutant of ASH1 that had been transformed with the vector control showed no ASH1 mRNA signal (Fig. 1A, bottom row). Uninucleate unbudded cells also showed ASH1 mRNA localization in an arc at the periphery on the cell. The unbudded cells most likely reflect cells that have recently finished cytokinesis.

Figure 1

Localization of ASH1 mRNA and Ash1p to daughter cells of budding yeast. Bars in Nomarski (Nom.) images in all figures represent 10 μM. (A) Simultaneous detection ofASH1 mRNA and poly(A)+ RNA by FISH (23). (B) Simultaneous detection ofASH1 mRNA and Ash1p-myc9 protein (24). DAPI, 4′,6′-diamidino-2-phenylindole.

To determine if localization of ASH1 mRNA to the bud tip resulted in higher rates of Ash1p synthesis in daughter cells, we simultaneously monitored the accumulation of ASH1 mRNA and Ash1p produced by an epitope-tagged ASH1 gene integrated at the ASH1 locus (Fig. 1B). Ash1p accumulated within the daughter nucleus near the ASH1 mRNA, which appeared to be “tracking” through the bud neck to the tip.

The she mutants that do not localize Ash1p asymmetrically (3) might do so by mislocalizing ASH1 mRNA. Analysis of the she mutants revealed that ASH1mRNA was present in binucleate or unbudded uninucleate cells but that it was no longer asymmetrically distributed (Fig.2). In myo4, she2, she3, andshe4 mutants, ASH1 mRNA was commonly distributed in a filamentous-like fashion or in patches at the cell periphery. In contrast, in she5/bni1 mutants, ASH1 mRNA was mainly concentrated in patches near the bud neck.

Figure 2

Dependence of localized ASH1 mRNA onSHE1, SHE2, SHE3, SHE4, and SHE5. Fractions of cells with localizedASH1 mRNA are as follows: K6278 [wild type (WT)], 0.50; K5205 (she5::URA3), <0.01; K5209 (she1::URA3), 0.01; K5235 (she3::URA3), 0.01; K5547 (she2::URA3), 0.01; and K5560 (she4::URA3), <0.01.

The dependence of ASH1 mRNA localization on the nonessential type V myosin Myo4 suggests that the actin cytoskeleton might participate in the asymmetric accumulation of ASH1 mRNA and protein. We therefore analyzed the distribution of ASH1 mRNA and Ash1p in strains carrying mutations that affect actin function. Theact1-133 temperature-sensitive mutation affects the myosin-binding site on actin and prevents bud formation at the restrictive temperature (7). In mutant cells growing at the permissive temperature, ASH1 mRNA was rarely localized to the bud (Fig. 3A), and Ash1p accumulation in binucleate cells was rarely asymmetric (8). Although theASH1 mRNA was diffusely localized in this mutant, the daughter cell often contained higher amounts of mRNA than the mother cell.

Figure 3

Mislocalization ofASH1 mRNA in strains defective in the actin cytoskeleton. (A) ASH1 mRNA distribution in strains K5985 (act1-133::HIS3) and K5986 (ACT1::HIS3). Each strain was transformed with plasmid C3431 (25) grown at 24°C to midlogarithmic phase in synthetic media minus uracil (18). A portion of each culture was maintained at 24°C, while a second portion of the culture was shifted to 31°C for 90 min. Fractions of cells with localizedASH1 mRNA are given in parentheses in the following: Strain K5985 was grown at 24°C (0.02) and shifted to 31°C (0.02), and strain K5986 was grown at 24°C (0.36) and shifted to 31°C (0.45). (B) ASH1 mRNA distribution in strains K5552 (wild type), K5917 (tpm1Δ::LEU2), and K5962 (pfy1-111::LEU2) (23). Each strain was transformed with plasmid C3431 and grown at 24°C. The fractions of cells in each strain with localized ASH1 mRNA are as follows: K5552, 0.51; K5917, 0.04; and K5962, 0.04. (C) ASH1 mRNA distribution in strains K5552 (wild type) and NY1006 (myo2-66) (10). Each strain was transformed with plasmid C3319 and grown as described above for the act1-133 strain.

We also investigated the distribution of ASH1 mRNA and Ash1p in mutants lacking the TPM1 gene that codes for the major tropomyosin isotype in yeast or in mutants that are defective in the profilin gene (PFY1) (Fig. 3B) (9).ASH1 mRNA was not localized in either mutant but rather had a distribution similar to that observed in the she1toshe4 mutants. Ash1p accumulation was rarely asymmetric in binucleate cells (8). All three mutants (act1, tpm1, and pfy1) affect the formation of actin cables that run from mother cells into their buds (7, 9).

The defective ASH1 mRNA and protein localization in these mutants was not an indirect effect of the perturbation in bud formation. Mutants carrying a temperature-sensitive allele ofmyo2, which encodes a type V myosin that is essential for bud formation (10), showed asymmetric localization ofASH1 mRNA and protein at both permissive and restrictive temperatures (Fig. 3C).

To determine if the ASH1 mRNA at the bud tip was transported through the bud neck and to test whether microtubules are involved in the transport, we analyzed the distribution of ASH1 mRNA in a tub2-401 mutant strain, which is defective in the formation of astral microtubules needed for anaphase to take place at the bud neck (11). Nuclear division occurs entirely within mother cells when this mutant is shifted to the restrictive temperature (18°C). The ASH1 mRNA was still localized to the distal bud tip even when nuclear division had occurred within the mother cell (Fig. 4A), indicating that the cells can transport ASH1 mRNA from the mother cell to the bud tip, despite disruption of microtubules.

Figure 4

Transport of ASH1 mRNA to daughter cells independent of the stage of the cell cycle. (A) Localization of ASH1 mRNA in atub2-401 mutant strain determined by FISH. Strain K5429 (tub2-401) was transformed with plasmid C3431. Transformants were grown to midlogarithmic phase at the permissive temperature (30°C), and a portion of the culture was shifted to the restrictive temperature (18°C) for 90 min. A second portion of the culture was maintained at 30°C for 90 min. Note at the nonpermissive temperature (18°C) the presence of two nuclei in mother cells, as shown by DAPI staining. (B) ASH1 mRNA localization when expressed from a galactose inducible promoter. Strain K6278 was transformed with plasmid C3348, a derivative of YCplac133 that encodes ASH1-myc9 under the control of the galactose-inducible promoter GAL1 (17). Transformants were grown to midlogarithmic phase in synthetic liquid media (minus uracil) containing either 2% glucose (repressing conditions) or 3% galactose (inducing conditions).

The question of whether the ASH1 mRNA is transported from mother cell nuclei to bud tips was also addressed by experiments in which ASH1 mRNA was expressed at a stage when there was only a single nucleus. In these experiments, ASH1 mRNA was expressed from the promoter for the GAL1 andGAL10 genes, which is inducible by the addition of galactose to the medium (12). The ASH1 mRNA localized to the bud at all stages of the cell cycle, from the beginning of bud formation during late G1 to the end of anaphase whenASH1 mRNA is normally produced; however, no ASH1mRNA was detectable when its expression was repressed by glucose (Fig.4B). This result demonstrates that (i) the mRNA localization mechanism is functional throughout most of the cell cycle; (ii) the mRNAs that are transported to the bud tip include those made within mother cell nuclei; and (iii) the factors at the bud tip that are responsible for attracting or anchoring ASH1 mRNA are there from the onset of bud formation.

In higher eukaryotic cells, cis-acting sequences that are necessary for mRNA localization are often confined to the 3′-untranslated region (3′-UTR) of the mRNA (4, 13). To address whetherASH1 mRNA contains a localization signal in its 3′-UTR, a hybrid mRNA was constructed and expressed in yeast. TheEscherichia coli lacZ coding sequence was fused to 250 nucleotides of ASH1 3′-UTR, and the resultant hybrid mRNA expressed from the GAL1 promoter was detected by FISH. Localization of the hybrid mRNA to the bud in uninucleate small budded cells and to daughters in binucleate budded cells was dependent on theASH1 3′-UTR (Fig. 5) and did not occur when the 3′-UTR from another mRNA (ADHII) was substituted (Fig. 5). The localization to buds of the hybrid mRNA containing the ASH1 3′-UTR was dependent on SHE1, because she1 mutant cells no longer localize the hybrid mRNA (Fig. 5). Thus, the 3′-UTR of the ASH1 mRNA appears to contain a cis-acting sequence element that is sufficient to target a heterologous mRNA to the bud. However, the ASH1 mRNA may contain redundant cis-acting factors that are responsible forASH1 mRNA localization, because replacement of the 3′-UTR ofASH1 with that of CDC6 only reducedASH1 mRNA localization from 56 to 40% and Ash1p asymmetry from 92 to 82%.

Figure 5

Localization of a heterologous mRNA to daughter cells through the 3′-UTR of ASH1. Strain K6278 was transformed with either plasmid pHZ18-polyadenylate [poly(A)] (5) or pXMRS25 (26). Plasmid pHZ18-poly(A) contains the lacZ reporter gene with the ADHII3′-UTR, whereas pXMRS25 contains the 3′-UTR of ASH1 in place of ADHII sequences. In situ hybridizations tolacZ were performed as described previously with the described modifications (5, 23).

The data presented here suggest that localization of ASH1mRNA is responsible for the asymmetry in mating type switching through Ash1p. Microscopically, we have shown the juxtaposition of the mRNA in the cytoplasm and the protein in the nucleus (Fig. 1B). Additionally, we have shown that mutations in eight different genes, which cause symmetrical Ash1p accumulation, also abolish localization ofASH1 mRNA. These observations strongly suggest thatASH1 mRNA localization is necessary for protein asymmetry. It is unlikely that all these mutations affect independently both mRNA localization and protein asymmetry, especially as some of the mutations (myo4, she2, and she3) are not very pleiotropic.

Cytoplasmic microtubules are thought to have a major role in mRNA localization both in Drosophila and Xenopusoocytes (4). However, actin has been shown to be the filament system that is important for the localization of β-actin mRNA in fibroblasts (14). Microtubules are probably not involved in ASH1 mRNA localization, because disruption of astral microtubules by the tub2-401 mutation had little or no effect, even though it did prevent nuclei from migrating to the bud neck. Instead, we found that mutations that affect what is presumably an actin-dependent motor protein, Myo4p, as well as mutations in tropomyosin, profilin, and actin itself, all greatly reduce or even abolish ASH1 mRNA localization to the distal tip. Both profilin and cytoplasmic tropomyosin have been implicated in localizingoskar mRNA to the posterior pole of Drosophilaoocytes (15).

We have shown that a mechanism that is capable of movingASH1 mRNA to the bud tip exists long before ASH1mRNA is actually made in the cell. Possibly, the true cargo for Myo4p is not ASH1 mRNA itself but some bud tip–specific protein to which ASH1 mRNA stably binds. Such hypothetical proteins have also been postulated to be the determinants of budding axis in diploid cells (16). ASH1 mRNA, therefore, might not be actively transported at all but merely bound to a receptor, which had previously been delivered to the daughter cell cortex by Myo4p. Irrespective of the actual mechanism by which ASH1mRNA is localized, it would seem likely that proteins localized to the distal bud tip act either as addresses or anchors for ASH1mRNA. Curiously, none of the proteins currently implicated inASH1 mRNA localization are known to be localized at the bud tip at the end of anaphase. This leads us to suspect that as yet unknown components of the localization mechanism await discovery. Likewise, it is probable that other yeast mRNA will be found to be localized to the bud tip or neck.

In multicellular organisms, most localized mRNAs code for proteins involved with differential gene expression. Our data show that asymmetric intracellular mRNA localization is not confined to cells from the animal kingdom or to those from multicellular organisms (4, 13). In this light, mRNA localization might be seen as a precursor to, or a substitute for, differential gene expression to generate spatial complexity of proteins.

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