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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

m6A accumulates prior to meiotic divisions.A) Western analysis for 3x-myc-tagged Ime4 protein (SAy914) throughout meiosis; Pgk1 protein serves as loading control. B) Quantification of m6A abundance relative to cytosine throughout meiosis (green triangles, left axis) in a wild-type strain (SAy821). Percent of 4C cells as quantified by FACS (3×104 cells/time point—blue diamonds, right axis) and percent cells undergoing nuclear divisions as assayed by DAPI staining (200 cells/time point—red squares, right axis) are shown as references for meiotic progression. C) Strand-specific qPCR for sense IME4 (red squares, left axis) and antisense transcript (IME4-as) (blue diamonds, right axis) transcript throughout meiosis. D) m6A relative to cytosine quantification in cells carrying an estradiol-inducible NDT80 construct as their sole source of NDT80 (SAy995). Cells were treated with β-estradiol or vehicle 6 hours after meiotic induction and monitored at 9 hours.
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pgen-1002732-g004: m6A accumulates prior to meiotic divisions.A) Western analysis for 3x-myc-tagged Ime4 protein (SAy914) throughout meiosis; Pgk1 protein serves as loading control. B) Quantification of m6A abundance relative to cytosine throughout meiosis (green triangles, left axis) in a wild-type strain (SAy821). Percent of 4C cells as quantified by FACS (3×104 cells/time point—blue diamonds, right axis) and percent cells undergoing nuclear divisions as assayed by DAPI staining (200 cells/time point—red squares, right axis) are shown as references for meiotic progression. C) Strand-specific qPCR for sense IME4 (red squares, left axis) and antisense transcript (IME4-as) (blue diamonds, right axis) transcript throughout meiosis. D) m6A relative to cytosine quantification in cells carrying an estradiol-inducible NDT80 construct as their sole source of NDT80 (SAy995). Cells were treated with β-estradiol or vehicle 6 hours after meiotic induction and monitored at 9 hours.

Mentions: To determine the period during starvation when Ime4 is most abundant, we analyzed Ime4 protein levels by Western blotting in a synchronous time course after inoculation in SPO. As shown in Figure 4A, Ime4 levels increased soon after the shift to starvation conditions and peaked during pre-meiotic S and G2/prophase. Once cells entered into the meiotic divisions, full-length Ime4 disappeared rapidly. We also observed the accumulation of a faster-migrating band that may represent a carboxy-terminal cleavage product of Ime4 (Figure 4A).


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

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

m6A accumulates prior to meiotic divisions.A) Western analysis for 3x-myc-tagged Ime4 protein (SAy914) throughout meiosis; Pgk1 protein serves as loading control. B) Quantification of m6A abundance relative to cytosine throughout meiosis (green triangles, left axis) in a wild-type strain (SAy821). Percent of 4C cells as quantified by FACS (3×104 cells/time point—blue diamonds, right axis) and percent cells undergoing nuclear divisions as assayed by DAPI staining (200 cells/time point—red squares, right axis) are shown as references for meiotic progression. C) Strand-specific qPCR for sense IME4 (red squares, left axis) and antisense transcript (IME4-as) (blue diamonds, right axis) transcript throughout meiosis. D) m6A relative to cytosine quantification in cells carrying an estradiol-inducible NDT80 construct as their sole source of NDT80 (SAy995). Cells were treated with β-estradiol or vehicle 6 hours after meiotic induction and monitored at 9 hours.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369947&req=5

pgen-1002732-g004: m6A accumulates prior to meiotic divisions.A) Western analysis for 3x-myc-tagged Ime4 protein (SAy914) throughout meiosis; Pgk1 protein serves as loading control. B) Quantification of m6A abundance relative to cytosine throughout meiosis (green triangles, left axis) in a wild-type strain (SAy821). Percent of 4C cells as quantified by FACS (3×104 cells/time point—blue diamonds, right axis) and percent cells undergoing nuclear divisions as assayed by DAPI staining (200 cells/time point—red squares, right axis) are shown as references for meiotic progression. C) Strand-specific qPCR for sense IME4 (red squares, left axis) and antisense transcript (IME4-as) (blue diamonds, right axis) transcript throughout meiosis. D) m6A relative to cytosine quantification in cells carrying an estradiol-inducible NDT80 construct as their sole source of NDT80 (SAy995). Cells were treated with β-estradiol or vehicle 6 hours after meiotic induction and monitored at 9 hours.
Mentions: To determine the period during starvation when Ime4 is most abundant, we analyzed Ime4 protein levels by Western blotting in a synchronous time course after inoculation in SPO. As shown in Figure 4A, Ime4 levels increased soon after the shift to starvation conditions and peaked during pre-meiotic S and G2/prophase. Once cells entered into the meiotic divisions, full-length Ime4 disappeared rapidly. We also observed the accumulation of a faster-migrating band that may represent a carboxy-terminal cleavage product of Ime4 (Figure 4A).

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