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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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Related in: MedlinePlus

RTG from starvation results in three distinct cell morphologies.(A) Representative morphologies of daughter cells following RTG from SPO throughout a meiotic time course. Top left panel: RTG at 0 hours, i.e., after growth in BYTA, (RTG0); top right panel: RTG at 3 hours (RTG3), which is comparable to PH cells from solid nitrogen medium (bottom left panel). Bottom right panel: RTG at 6 hours (RTG6). RTG0 and RTG3 cells were photographed after one complete cell cycle, corresponding to 160 minutes and 220 minutes after shift to rich medium, respectively (see Figure S1A). RTG6 cells were photographed three hours after shift to rich medium, at which point the majority of cells (>95%) had formed spores. Arrows indicate primary daughter cells upon RTG0 and RTG3. B) Quantification of axial ratio in wild-type (SAy821) cells upon RTG throughout a meiotic time course (red bars, left axis; n = 200 cells/time point) relative to percent of cells undergoing pre-meiotic DNA synthesis (i.e., 4C cells) (blue diamonds, right axis, quantified by FACS, 3×104 cells/time point) and percent cells undergoing meiotic divisions as assayed by DAPI staining (green triangles, right axis). Schematic at top defines axial ratio. The majority (>80%) of RTG5, RTG6, RTG7, and RTG9 cells either formed spores or remained unbudded three hours after shift to rich medium; axial ratio was therefore not quantified for these time points. C) MATa/α diploids (SAy821) (top left panel) or MAT α haploids (H224) (top right panel) were returned to growth in rich medium after meiotic induction. Arrows indicate primary buds. Bottom panels represent colony morphologies after growth on SLAD for 6 days. D) Distribution of axial ratios of primary daughter cells upon RTG3 for strains in (C): wild-type diploid (SAy821 top panel), haploid MAT α (H224 bottom panel) (n = 200 cells/strain). RTG0 is represented in red bars, RTG3 in blue bars. E) Wild-type (SAy821), flo11Δ/Δ (SAy789) and flo8Δ/Δ (SAy905) daughter cell morphologies upon RTG3 (top panels). Arrows indicate primary daughter cells. The same strains were photographed after growth on SLAD for 6 days (bottom panels). Axial ratios are quantified in (F) (n = 200 cells/strain).
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pgen-1002732-g001: RTG from starvation results in three distinct cell morphologies.(A) Representative morphologies of daughter cells following RTG from SPO throughout a meiotic time course. Top left panel: RTG at 0 hours, i.e., after growth in BYTA, (RTG0); top right panel: RTG at 3 hours (RTG3), which is comparable to PH cells from solid nitrogen medium (bottom left panel). Bottom right panel: RTG at 6 hours (RTG6). RTG0 and RTG3 cells were photographed after one complete cell cycle, corresponding to 160 minutes and 220 minutes after shift to rich medium, respectively (see Figure S1A). RTG6 cells were photographed three hours after shift to rich medium, at which point the majority of cells (>95%) had formed spores. Arrows indicate primary daughter cells upon RTG0 and RTG3. B) Quantification of axial ratio in wild-type (SAy821) cells upon RTG throughout a meiotic time course (red bars, left axis; n = 200 cells/time point) relative to percent of cells undergoing pre-meiotic DNA synthesis (i.e., 4C cells) (blue diamonds, right axis, quantified by FACS, 3×104 cells/time point) and percent cells undergoing meiotic divisions as assayed by DAPI staining (green triangles, right axis). Schematic at top defines axial ratio. The majority (>80%) of RTG5, RTG6, RTG7, and RTG9 cells either formed spores or remained unbudded three hours after shift to rich medium; axial ratio was therefore not quantified for these time points. C) MATa/α diploids (SAy821) (top left panel) or MAT α haploids (H224) (top right panel) were returned to growth in rich medium after meiotic induction. Arrows indicate primary buds. Bottom panels represent colony morphologies after growth on SLAD for 6 days. D) Distribution of axial ratios of primary daughter cells upon RTG3 for strains in (C): wild-type diploid (SAy821 top panel), haploid MAT α (H224 bottom panel) (n = 200 cells/strain). RTG0 is represented in red bars, RTG3 in blue bars. E) Wild-type (SAy821), flo11Δ/Δ (SAy789) and flo8Δ/Δ (SAy905) daughter cell morphologies upon RTG3 (top panels). Arrows indicate primary daughter cells. The same strains were photographed after growth on SLAD for 6 days (bottom panels). Axial ratios are quantified in (F) (n = 200 cells/strain).

Mentions: We investigated the relationship between meiotic induction and PH growth in SK1, a strain that efficiently undergoes both the PH and meiotic developmental programs [9], [20]. To analyze the early starvation response in a controlled and synchronous manner, we employed a Return-To-Growth (RTG) assay [19], [21], in which diploid cells were incubated for a given amount of time in extremely nutrient-poor liquid medium (SPO) before being returned to rich medium (YPD). Previous studies used this approach to show that cells exiting from meiotic G2/prophase are committed to meiosis and will complete sporulation even in rich medium [22], [23], suggesting that analysis of cell morphology after RTG provides a measure for the developmental potential of nutrient-starved cells. By analyzing morphological changes at hourly intervals in an RTG time course, we confirmed that cells become committed to meiosis as they exit from meiotic G2/prophase and enter the meiotic divisions. This commitment occurred 5 to 7 hours after inoculation in SPO in our strains (Figure 1A, 1B).


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

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

RTG from starvation results in three distinct cell morphologies.(A) Representative morphologies of daughter cells following RTG from SPO throughout a meiotic time course. Top left panel: RTG at 0 hours, i.e., after growth in BYTA, (RTG0); top right panel: RTG at 3 hours (RTG3), which is comparable to PH cells from solid nitrogen medium (bottom left panel). Bottom right panel: RTG at 6 hours (RTG6). RTG0 and RTG3 cells were photographed after one complete cell cycle, corresponding to 160 minutes and 220 minutes after shift to rich medium, respectively (see Figure S1A). RTG6 cells were photographed three hours after shift to rich medium, at which point the majority of cells (>95%) had formed spores. Arrows indicate primary daughter cells upon RTG0 and RTG3. B) Quantification of axial ratio in wild-type (SAy821) cells upon RTG throughout a meiotic time course (red bars, left axis; n = 200 cells/time point) relative to percent of cells undergoing pre-meiotic DNA synthesis (i.e., 4C cells) (blue diamonds, right axis, quantified by FACS, 3×104 cells/time point) and percent cells undergoing meiotic divisions as assayed by DAPI staining (green triangles, right axis). Schematic at top defines axial ratio. The majority (>80%) of RTG5, RTG6, RTG7, and RTG9 cells either formed spores or remained unbudded three hours after shift to rich medium; axial ratio was therefore not quantified for these time points. C) MATa/α diploids (SAy821) (top left panel) or MAT α haploids (H224) (top right panel) were returned to growth in rich medium after meiotic induction. Arrows indicate primary buds. Bottom panels represent colony morphologies after growth on SLAD for 6 days. D) Distribution of axial ratios of primary daughter cells upon RTG3 for strains in (C): wild-type diploid (SAy821 top panel), haploid MAT α (H224 bottom panel) (n = 200 cells/strain). RTG0 is represented in red bars, RTG3 in blue bars. E) Wild-type (SAy821), flo11Δ/Δ (SAy789) and flo8Δ/Δ (SAy905) daughter cell morphologies upon RTG3 (top panels). Arrows indicate primary daughter cells. The same strains were photographed after growth on SLAD for 6 days (bottom panels). Axial ratios are quantified in (F) (n = 200 cells/strain).
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Related In: Results  -  Collection

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pgen-1002732-g001: RTG from starvation results in three distinct cell morphologies.(A) Representative morphologies of daughter cells following RTG from SPO throughout a meiotic time course. Top left panel: RTG at 0 hours, i.e., after growth in BYTA, (RTG0); top right panel: RTG at 3 hours (RTG3), which is comparable to PH cells from solid nitrogen medium (bottom left panel). Bottom right panel: RTG at 6 hours (RTG6). RTG0 and RTG3 cells were photographed after one complete cell cycle, corresponding to 160 minutes and 220 minutes after shift to rich medium, respectively (see Figure S1A). RTG6 cells were photographed three hours after shift to rich medium, at which point the majority of cells (>95%) had formed spores. Arrows indicate primary daughter cells upon RTG0 and RTG3. B) Quantification of axial ratio in wild-type (SAy821) cells upon RTG throughout a meiotic time course (red bars, left axis; n = 200 cells/time point) relative to percent of cells undergoing pre-meiotic DNA synthesis (i.e., 4C cells) (blue diamonds, right axis, quantified by FACS, 3×104 cells/time point) and percent cells undergoing meiotic divisions as assayed by DAPI staining (green triangles, right axis). Schematic at top defines axial ratio. The majority (>80%) of RTG5, RTG6, RTG7, and RTG9 cells either formed spores or remained unbudded three hours after shift to rich medium; axial ratio was therefore not quantified for these time points. C) MATa/α diploids (SAy821) (top left panel) or MAT α haploids (H224) (top right panel) were returned to growth in rich medium after meiotic induction. Arrows indicate primary buds. Bottom panels represent colony morphologies after growth on SLAD for 6 days. D) Distribution of axial ratios of primary daughter cells upon RTG3 for strains in (C): wild-type diploid (SAy821 top panel), haploid MAT α (H224 bottom panel) (n = 200 cells/strain). RTG0 is represented in red bars, RTG3 in blue bars. E) Wild-type (SAy821), flo11Δ/Δ (SAy789) and flo8Δ/Δ (SAy905) daughter cell morphologies upon RTG3 (top panels). Arrows indicate primary daughter cells. The same strains were photographed after growth on SLAD for 6 days (bottom panels). Axial ratios are quantified in (F) (n = 200 cells/strain).
Mentions: We investigated the relationship between meiotic induction and PH growth in SK1, a strain that efficiently undergoes both the PH and meiotic developmental programs [9], [20]. To analyze the early starvation response in a controlled and synchronous manner, we employed a Return-To-Growth (RTG) assay [19], [21], in which diploid cells were incubated for a given amount of time in extremely nutrient-poor liquid medium (SPO) before being returned to rich medium (YPD). Previous studies used this approach to show that cells exiting from meiotic G2/prophase are committed to meiosis and will complete sporulation even in rich medium [22], [23], suggesting that analysis of cell morphology after RTG provides a measure for the developmental potential of nutrient-starved cells. By analyzing morphological changes at hourly intervals in an RTG time course, we confirmed that cells become committed to meiosis as they exit from meiotic G2/prophase and enter the meiotic divisions. This commitment occurred 5 to 7 hours after inoculation in SPO in our strains (Figure 1A, 1B).

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