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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

MIS complex expression is sufficient to induce m6A accumulation on mRNA.A) m6A accumulation on mRNA was quantified in rich conditions in wild-type (SAy821), ime4Δ/Δ (SAy771), PCUP1-IME4 (SAy1249), PCUP1-MUM2 (SAy1251), PCUP1-SLZ1 (SAy1250), PCUP1-IME4 PCUP1-MUM2 PCUP1-SLZ1 (SAy1248) and PCUP1-IME4 PCUP1-IME4 (SAy1252) after 150 minutes of mitotic growth in the presence of cupric sulfate. B) Western analysis for expression of Ime4 and Mum2. Cells encoding epitope-tagged Ime4 and Mum2 (SAy1232) were collected either from the rich cupric-sulfate media conditions in (A) (first column) or at 0, 2, and 5 hours in meiosis (as labeled), then subjected to Western analysis for either Ime4 (anti-myc) or Mum2 (anti-HA). Pgk1 serves as a loading control. Images across each row come from the same exposure.
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pgen-1002732-g006: MIS complex expression is sufficient to induce m6A accumulation on mRNA.A) m6A accumulation on mRNA was quantified in rich conditions in wild-type (SAy821), ime4Δ/Δ (SAy771), PCUP1-IME4 (SAy1249), PCUP1-MUM2 (SAy1251), PCUP1-SLZ1 (SAy1250), PCUP1-IME4 PCUP1-MUM2 PCUP1-SLZ1 (SAy1248) and PCUP1-IME4 PCUP1-IME4 (SAy1252) after 150 minutes of mitotic growth in the presence of cupric sulfate. B) Western analysis for expression of Ime4 and Mum2. Cells encoding epitope-tagged Ime4 and Mum2 (SAy1232) were collected either from the rich cupric-sulfate media conditions in (A) (first column) or at 0, 2, and 5 hours in meiosis (as labeled), then subjected to Western analysis for either Ime4 (anti-myc) or Mum2 (anti-HA). Pgk1 serves as a loading control. Images across each row come from the same exposure.

Mentions: The necessity of IME4, MUM2, and SLZ1 for the methylation of mRNA during meiosis raised the question of whether expression of these genes was sufficient to induce the methylation of mRNA. To test this, one copy of each gene was placed under control of the inducible CUP1 promoter in diploid cells while the other copy remained unaltered. Expression of these genes was induced by the addition of cupric sulfate in rich medium, a condition in which m6A does not normally accumulate on mRNA (Figure 6A). Under these growth conditions, neither Mum2 nor Ime4 were expressed at levels close to those found in meiosis (Figure 6B); Slz1 did not accumulate in cells until induction of meiotic development (Figure 5B). We found that inducing expression of IME4, MUM2, or SLZ1 singly in these conditions was not sufficient to induce m6A accumulation on mRNA (Figure 6A). By contrast, induction of both IME4 and MUM2 resulted in a strong accumulation of m6A. Induction of all three MIS components (MUM2, IME4, and SLZ1) further elevated m6A, albeit only by a small fraction, consistent with the role of SLZ1 as a non-essential component of the MIS complex (Figure 6A). Notably, none of the strains that express m6A in rich conditions exhibited any obvious morphological or growth differences as compared to un-induced control cells (data not shown). These data suggest that the restriction of mRNA methylation to times of starvation is largely a result of the starvation-specific expression of the MIS complex. Taken together, these phenotypes suggest a model in which Mum2 and Ime4 are essential components of an RNA methyltransferase complex, with Slz1 providing an accessory role necessary for optimal function.


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

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

MIS complex expression is sufficient to induce m6A accumulation on mRNA.A) m6A accumulation on mRNA was quantified in rich conditions in wild-type (SAy821), ime4Δ/Δ (SAy771), PCUP1-IME4 (SAy1249), PCUP1-MUM2 (SAy1251), PCUP1-SLZ1 (SAy1250), PCUP1-IME4 PCUP1-MUM2 PCUP1-SLZ1 (SAy1248) and PCUP1-IME4 PCUP1-IME4 (SAy1252) after 150 minutes of mitotic growth in the presence of cupric sulfate. B) Western analysis for expression of Ime4 and Mum2. Cells encoding epitope-tagged Ime4 and Mum2 (SAy1232) were collected either from the rich cupric-sulfate media conditions in (A) (first column) or at 0, 2, and 5 hours in meiosis (as labeled), then subjected to Western analysis for either Ime4 (anti-myc) or Mum2 (anti-HA). Pgk1 serves as a loading control. Images across each row come from the same exposure.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002732-g006: MIS complex expression is sufficient to induce m6A accumulation on mRNA.A) m6A accumulation on mRNA was quantified in rich conditions in wild-type (SAy821), ime4Δ/Δ (SAy771), PCUP1-IME4 (SAy1249), PCUP1-MUM2 (SAy1251), PCUP1-SLZ1 (SAy1250), PCUP1-IME4 PCUP1-MUM2 PCUP1-SLZ1 (SAy1248) and PCUP1-IME4 PCUP1-IME4 (SAy1252) after 150 minutes of mitotic growth in the presence of cupric sulfate. B) Western analysis for expression of Ime4 and Mum2. Cells encoding epitope-tagged Ime4 and Mum2 (SAy1232) were collected either from the rich cupric-sulfate media conditions in (A) (first column) or at 0, 2, and 5 hours in meiosis (as labeled), then subjected to Western analysis for either Ime4 (anti-myc) or Mum2 (anti-HA). Pgk1 serves as a loading control. Images across each row come from the same exposure.
Mentions: The necessity of IME4, MUM2, and SLZ1 for the methylation of mRNA during meiosis raised the question of whether expression of these genes was sufficient to induce the methylation of mRNA. To test this, one copy of each gene was placed under control of the inducible CUP1 promoter in diploid cells while the other copy remained unaltered. Expression of these genes was induced by the addition of cupric sulfate in rich medium, a condition in which m6A does not normally accumulate on mRNA (Figure 6A). Under these growth conditions, neither Mum2 nor Ime4 were expressed at levels close to those found in meiosis (Figure 6B); Slz1 did not accumulate in cells until induction of meiotic development (Figure 5B). We found that inducing expression of IME4, MUM2, or SLZ1 singly in these conditions was not sufficient to induce m6A accumulation on mRNA (Figure 6A). By contrast, induction of both IME4 and MUM2 resulted in a strong accumulation of m6A. Induction of all three MIS components (MUM2, IME4, and SLZ1) further elevated m6A, albeit only by a small fraction, consistent with the role of SLZ1 as a non-essential component of the MIS complex (Figure 6A). Notably, none of the strains that express m6A in rich conditions exhibited any obvious morphological or growth differences as compared to un-induced control cells (data not shown). These data suggest that the restriction of mRNA methylation to times of starvation is largely a result of the starvation-specific expression of the MIS complex. Taken together, these phenotypes suggest a model in which Mum2 and Ime4 are essential components of an RNA methyltransferase complex, with Slz1 providing an accessory role necessary for optimal function.

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