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RNA methylation by the MIS complex regulates a cell fate decision in yeast.

Agarwala SD, Blitzblau HG, Hochwagen A, Fink GR - PLoS Genet. (2012)

Bottom Line: Normal levels of meiotic mRNA methylation required the catalytic domain of Ime4, as well as two meiotic proteins, Mum2 and Slz1, which interacted and co-immunoprecipitated with Ime4.This MIS complex (Mum2, Ime4, and Slz1) functioned in both starvation pathways.Together, our results support the notion that the yeast starvation response is an extended process that progressively restricts cell fate and reveal a broad role of post-transcriptional RNA methylation in these decisions.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute, Cambridge, Massachusetts, USA.

ABSTRACT
For the yeast Saccharomyces cerevisiae, nutrient limitation is a key developmental signal causing diploid cells to switch from yeast-form budding to either foraging pseudohyphal (PH) growth or meiosis and sporulation. Prolonged starvation leads to lineage restriction, such that cells exiting meiotic prophase are committed to complete sporulation even if nutrients are restored. Here, we have identified an earlier commitment point in the starvation program. After this point, cells, returned to nutrient-rich medium, entered a form of synchronous PH development that was morphologically and genetically indistinguishable from starvation-induced PH growth. We show that lineage restriction during this time was, in part, dependent on the mRNA methyltransferase activity of Ime4, which played separable roles in meiotic induction and suppression of the PH program. Normal levels of meiotic mRNA methylation required the catalytic domain of Ime4, as well as two meiotic proteins, Mum2 and Slz1, which interacted and co-immunoprecipitated with Ime4. This MIS complex (Mum2, Ime4, and Slz1) functioned in both starvation pathways. Together, our results support the notion that the yeast starvation response is an extended process that progressively restricts cell fate and reveal a broad role of post-transcriptional RNA methylation in these decisions.

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Cells encoding IME4 mutant alleles show developmental defects.A) FACS analysis of DNA synthesis in wild-type (SAy821), ime4-cat/cat (SAy1086) and ime4Δ/Δ (SAy771) strains throughout a meiotic time course (n = 3×104 cells/strain/time point). DNA content of diploid cells before DNA replication (2C) and after DNA replication (4C) is indicated. B) NDT80 transcript levels in the strains from (A) during a meiotic time course. Transcript levels were determined by RT-PCR and normalized to ACT1 transcript levels. C) Kinetics for meiotic nuclear divisions as assayed by DAPI DNA staining in the strains from (A) (n = 200 cells/strain). D) Number of asci with one, two, three, four, or no spores in the strains from (A) after 24 hours in SPO medium (n = 200 cell/strain). E) Spore viability in the strains from (A); legend indicates number of surviving spores upon dissection (n = 187 tetrads/strain). F) Representative images of cells from strains in (A) after RTG3 (top panels—arrows indicate primary buds) and colonies grown on SLAD for 6 days (bottom panels). G) Quantification of axial ratios of RTG3 cells shown in (F), (n = 200 cells/strain). H) Representative images of cells after RTG3 (top panels—arrows indicate primary buds) and colonies grown on SLAD for 6 days (bottom panels) of wild-type (SAy821), ime2Δ/Δ (SAy859), ime4Δ/Δ (SAy771) or ime2Δ/Δ ime4Δ/Δ (SAy1123) strains. Arrows indicate primary daughter cells. I) Quantification of axial ratios of RTG3 cells shown in (H), (n = 200 cells/strain).
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pgen-1002732-g003: Cells encoding IME4 mutant alleles show developmental defects.A) FACS analysis of DNA synthesis in wild-type (SAy821), ime4-cat/cat (SAy1086) and ime4Δ/Δ (SAy771) strains throughout a meiotic time course (n = 3×104 cells/strain/time point). DNA content of diploid cells before DNA replication (2C) and after DNA replication (4C) is indicated. B) NDT80 transcript levels in the strains from (A) during a meiotic time course. Transcript levels were determined by RT-PCR and normalized to ACT1 transcript levels. C) Kinetics for meiotic nuclear divisions as assayed by DAPI DNA staining in the strains from (A) (n = 200 cells/strain). D) Number of asci with one, two, three, four, or no spores in the strains from (A) after 24 hours in SPO medium (n = 200 cell/strain). E) Spore viability in the strains from (A); legend indicates number of surviving spores upon dissection (n = 187 tetrads/strain). F) Representative images of cells from strains in (A) after RTG3 (top panels—arrows indicate primary buds) and colonies grown on SLAD for 6 days (bottom panels). G) Quantification of axial ratios of RTG3 cells shown in (F), (n = 200 cells/strain). H) Representative images of cells after RTG3 (top panels—arrows indicate primary buds) and colonies grown on SLAD for 6 days (bottom panels) of wild-type (SAy821), ime2Δ/Δ (SAy859), ime4Δ/Δ (SAy771) or ime2Δ/Δ ime4Δ/Δ (SAy1123) strains. Arrows indicate primary daughter cells. I) Quantification of axial ratios of RTG3 cells shown in (H), (n = 200 cells/strain).

Mentions: Because IME1 and IME2 are both regulated by the RNA methyltransferase Ime4, we also analyzed meiosis and RTG-PH development in ime4Δ/Δ mutants. Previous work demonstrated that IME4 promotes efficient entry into meiosis, although the severity of the meiotic entry defect of ime4Δ/Δ mutants varies considerably between strain backgrounds [17], [18]. In SK1, ime4Δ/Δ mutants exhibited only a minor delay in the initiation of meiotic DNA replication (Figure 3A), but were severely delayed in exit from meiotic G2/prophase as determined by monitoring the induction of the middle-meiotic transcription factor NDT80 and the kinetics of meiotic DNA segregation (Figure 3B, 3C). Additionally, spore formation was substantially reduced in ime4Δ/Δ cells (Figure 3D). However, the viability of those spores that formed was comparable to that of wild-type cells, indicating that, although the meiotic divisions were delayed, chromosome segregation was not affected in these strains (Figure 3E). A strain carrying point mutations in the evolutionarily-conserved catalytic motif IV of Ime4 (ime4-D349A, W351A—referred as ime4-cat) [12], [18] recapitulated these phenotypes, albeit with reduced severity (Figure 3A–3E), indicating that the RNA methyltransferase activity of Ime4 contributes to the efficient progression through meiosis.


RNA methylation by the MIS complex regulates a cell fate decision in yeast.

Agarwala SD, Blitzblau HG, Hochwagen A, Fink GR - PLoS Genet. (2012)

Cells encoding IME4 mutant alleles show developmental defects.A) FACS analysis of DNA synthesis in wild-type (SAy821), ime4-cat/cat (SAy1086) and ime4Δ/Δ (SAy771) strains throughout a meiotic time course (n = 3×104 cells/strain/time point). DNA content of diploid cells before DNA replication (2C) and after DNA replication (4C) is indicated. B) NDT80 transcript levels in the strains from (A) during a meiotic time course. Transcript levels were determined by RT-PCR and normalized to ACT1 transcript levels. C) Kinetics for meiotic nuclear divisions as assayed by DAPI DNA staining in the strains from (A) (n = 200 cells/strain). D) Number of asci with one, two, three, four, or no spores in the strains from (A) after 24 hours in SPO medium (n = 200 cell/strain). E) Spore viability in the strains from (A); legend indicates number of surviving spores upon dissection (n = 187 tetrads/strain). F) Representative images of cells from strains in (A) after RTG3 (top panels—arrows indicate primary buds) and colonies grown on SLAD for 6 days (bottom panels). G) Quantification of axial ratios of RTG3 cells shown in (F), (n = 200 cells/strain). H) Representative images of cells after RTG3 (top panels—arrows indicate primary buds) and colonies grown on SLAD for 6 days (bottom panels) of wild-type (SAy821), ime2Δ/Δ (SAy859), ime4Δ/Δ (SAy771) or ime2Δ/Δ ime4Δ/Δ (SAy1123) strains. Arrows indicate primary daughter cells. I) Quantification of axial ratios of RTG3 cells shown in (H), (n = 200 cells/strain).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pgen-1002732-g003: Cells encoding IME4 mutant alleles show developmental defects.A) FACS analysis of DNA synthesis in wild-type (SAy821), ime4-cat/cat (SAy1086) and ime4Δ/Δ (SAy771) strains throughout a meiotic time course (n = 3×104 cells/strain/time point). DNA content of diploid cells before DNA replication (2C) and after DNA replication (4C) is indicated. B) NDT80 transcript levels in the strains from (A) during a meiotic time course. Transcript levels were determined by RT-PCR and normalized to ACT1 transcript levels. C) Kinetics for meiotic nuclear divisions as assayed by DAPI DNA staining in the strains from (A) (n = 200 cells/strain). D) Number of asci with one, two, three, four, or no spores in the strains from (A) after 24 hours in SPO medium (n = 200 cell/strain). E) Spore viability in the strains from (A); legend indicates number of surviving spores upon dissection (n = 187 tetrads/strain). F) Representative images of cells from strains in (A) after RTG3 (top panels—arrows indicate primary buds) and colonies grown on SLAD for 6 days (bottom panels). G) Quantification of axial ratios of RTG3 cells shown in (F), (n = 200 cells/strain). H) Representative images of cells after RTG3 (top panels—arrows indicate primary buds) and colonies grown on SLAD for 6 days (bottom panels) of wild-type (SAy821), ime2Δ/Δ (SAy859), ime4Δ/Δ (SAy771) or ime2Δ/Δ ime4Δ/Δ (SAy1123) strains. Arrows indicate primary daughter cells. I) Quantification of axial ratios of RTG3 cells shown in (H), (n = 200 cells/strain).
Mentions: Because IME1 and IME2 are both regulated by the RNA methyltransferase Ime4, we also analyzed meiosis and RTG-PH development in ime4Δ/Δ mutants. Previous work demonstrated that IME4 promotes efficient entry into meiosis, although the severity of the meiotic entry defect of ime4Δ/Δ mutants varies considerably between strain backgrounds [17], [18]. In SK1, ime4Δ/Δ mutants exhibited only a minor delay in the initiation of meiotic DNA replication (Figure 3A), but were severely delayed in exit from meiotic G2/prophase as determined by monitoring the induction of the middle-meiotic transcription factor NDT80 and the kinetics of meiotic DNA segregation (Figure 3B, 3C). Additionally, spore formation was substantially reduced in ime4Δ/Δ cells (Figure 3D). However, the viability of those spores that formed was comparable to that of wild-type cells, indicating that, although the meiotic divisions were delayed, chromosome segregation was not affected in these strains (Figure 3E). A strain carrying point mutations in the evolutionarily-conserved catalytic motif IV of Ime4 (ime4-D349A, W351A—referred as ime4-cat) [12], [18] recapitulated these phenotypes, albeit with reduced severity (Figure 3A–3E), indicating that the RNA methyltransferase activity of Ime4 contributes to the efficient progression through meiosis.

Bottom Line: Normal levels of meiotic mRNA methylation required the catalytic domain of Ime4, as well as two meiotic proteins, Mum2 and Slz1, which interacted and co-immunoprecipitated with Ime4.This MIS complex (Mum2, Ime4, and Slz1) functioned in both starvation pathways.Together, our results support the notion that the yeast starvation response is an extended process that progressively restricts cell fate and reveal a broad role of post-transcriptional RNA methylation in these decisions.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute, Cambridge, Massachusetts, USA.

ABSTRACT
For the yeast Saccharomyces cerevisiae, nutrient limitation is a key developmental signal causing diploid cells to switch from yeast-form budding to either foraging pseudohyphal (PH) growth or meiosis and sporulation. Prolonged starvation leads to lineage restriction, such that cells exiting meiotic prophase are committed to complete sporulation even if nutrients are restored. Here, we have identified an earlier commitment point in the starvation program. After this point, cells, returned to nutrient-rich medium, entered a form of synchronous PH development that was morphologically and genetically indistinguishable from starvation-induced PH growth. We show that lineage restriction during this time was, in part, dependent on the mRNA methyltransferase activity of Ime4, which played separable roles in meiotic induction and suppression of the PH program. Normal levels of meiotic mRNA methylation required the catalytic domain of Ime4, as well as two meiotic proteins, Mum2 and Slz1, which interacted and co-immunoprecipitated with Ime4. This MIS complex (Mum2, Ime4, and Slz1) functioned in both starvation pathways. Together, our results support the notion that the yeast starvation response is an extended process that progressively restricts cell fate and reveal a broad role of post-transcriptional RNA methylation in these decisions.

Show MeSH
Related in: MedlinePlus