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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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m6A formation inhibits filamentation.A) Quantification of m6A abundance relative to cytosine (blue bars, left axis) and budding index (green triangles, right axis) upon RTG3. B) Western analysis for 3x-myc-tagged Ime4 protein (SAy914), 3x-HA-tagged Mum2 protein (SAy1235) or 3x-HA-tagged Slz1 protein (SAy1254) throughout RTG3 (i.e., following the shift to YPD after 3 hours in SPO); Pgk1 protein serves as loading control. C) Representative images of cells from wild-type (SAy821), ime4Δ/Δ (SAy771) and a strain induced to express the three components, IME4, MUM2 and SLZ1 (SAy1248) from PCUP1 after RTG3. All strains were treated with cupric sulfate upon RTG3 into YPD. D) Axial ratio quantifications of RTG3 cells from cells in (C) (n = 200 cells/strain).
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pgen-1002732-g007: m6A formation inhibits filamentation.A) Quantification of m6A abundance relative to cytosine (blue bars, left axis) and budding index (green triangles, right axis) upon RTG3. B) Western analysis for 3x-myc-tagged Ime4 protein (SAy914), 3x-HA-tagged Mum2 protein (SAy1235) or 3x-HA-tagged Slz1 protein (SAy1254) throughout RTG3 (i.e., following the shift to YPD after 3 hours in SPO); Pgk1 protein serves as loading control. C) Representative images of cells from wild-type (SAy821), ime4Δ/Δ (SAy771) and a strain induced to express the three components, IME4, MUM2 and SLZ1 (SAy1248) from PCUP1 after RTG3. All strains were treated with cupric sulfate upon RTG3 into YPD. D) Axial ratio quantifications of RTG3 cells from cells in (C) (n = 200 cells/strain).

Mentions: The MIS complex acts as an inhibitor of PH development, which raises the question of how cells are able to enter RTG-PH development during meiotic G2/prophase when the levels of MIS complex components and m6A are high. To answer this question, we monitored m6A levels upon RTG3. This analysis revealed that m6A levels dropped rapidly after RTG3, approaching the level of vegetative cells by 75 minutes after RTG3, shortly before bud formation was initiated in RTG cultures (Figure 7A). These observations suggest that MIS-dependent inhibition of PH development is alleviated in time to allow elongated bud growth in RTG3 cultures.


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 formation inhibits filamentation.A) Quantification of m6A abundance relative to cytosine (blue bars, left axis) and budding index (green triangles, right axis) upon RTG3. B) Western analysis for 3x-myc-tagged Ime4 protein (SAy914), 3x-HA-tagged Mum2 protein (SAy1235) or 3x-HA-tagged Slz1 protein (SAy1254) throughout RTG3 (i.e., following the shift to YPD after 3 hours in SPO); Pgk1 protein serves as loading control. C) Representative images of cells from wild-type (SAy821), ime4Δ/Δ (SAy771) and a strain induced to express the three components, IME4, MUM2 and SLZ1 (SAy1248) from PCUP1 after RTG3. All strains were treated with cupric sulfate upon RTG3 into YPD. D) Axial ratio quantifications of RTG3 cells from cells in (C) (n = 200 cells/strain).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002732-g007: m6A formation inhibits filamentation.A) Quantification of m6A abundance relative to cytosine (blue bars, left axis) and budding index (green triangles, right axis) upon RTG3. B) Western analysis for 3x-myc-tagged Ime4 protein (SAy914), 3x-HA-tagged Mum2 protein (SAy1235) or 3x-HA-tagged Slz1 protein (SAy1254) throughout RTG3 (i.e., following the shift to YPD after 3 hours in SPO); Pgk1 protein serves as loading control. C) Representative images of cells from wild-type (SAy821), ime4Δ/Δ (SAy771) and a strain induced to express the three components, IME4, MUM2 and SLZ1 (SAy1248) from PCUP1 after RTG3. All strains were treated with cupric sulfate upon RTG3 into YPD. D) Axial ratio quantifications of RTG3 cells from cells in (C) (n = 200 cells/strain).
Mentions: The MIS complex acts as an inhibitor of PH development, which raises the question of how cells are able to enter RTG-PH development during meiotic G2/prophase when the levels of MIS complex components and m6A are high. To answer this question, we monitored m6A levels upon RTG3. This analysis revealed that m6A levels dropped rapidly after RTG3, approaching the level of vegetative cells by 75 minutes after RTG3, shortly before bud formation was initiated in RTG cultures (Figure 7A). These observations suggest that MIS-dependent inhibition of PH development is alleviated in time to allow elongated bud growth in RTG3 cultures.

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