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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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Mum2 and Slz1 interact with Ime4 and are required for m6A formation.A) Western analysis for co-immunoprecipitation of Mum2 (left panels) and Slz1 (right panels) with Ime4. HA-tagged Mum2 or Slz1 was immunoprecipitated from cellular extracts 3 hours after induction of meiosis and probed for interaction with myc-tagged Ime4 (SAy1232, SAy1253, respectively). A myc-Ime4 (SAy914) strain without HA-tags served as a control. Arrows in the IP lanes indicate IgG bands. B) Western analysis for HA-tagged Mum2 protein (SAy1235) or HA-tagged Slz1 protein (SAy1254) throughout meiosis; Pgk1 protein serves as loading control. C) Quantification of m6A on mRNA three hours after meiotic starvation, when m6A accumulation is maximal in wild-type cells (SAy821). Deleting any one of ime4Δ/Δ (SAy771), mum2Δ/Δ (SAy1196) and slz1Δ/Δ (SAy1206) results in a reduction in m6A levels. D) Wild-type (SAy821), ime4Δ/Δ (SAy771), mum2Δ/Δ (SAy1196) and slz1Δ/Δ (SAy1206) daughter cell morphology upon RTG3 (top panels). Arrows indicate primary buds. The same strains were photographed after growth on SLAD for 6 days (bottom panels). E) Axial ratio quantification of primary daughter cells upon RTG3 for strains in (D). (F) FACS analysis of DNA synthesis in strains from (D) 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. G) Kinetics for meiotic nuclear divisions as assayed by DNA staining by DAPI in the strains from (C) (n = 200 cells/strain/time point). H) Number of asci with one, two, three/four, or no spores in strains from (D) after 24 hours in SPO medium (n = 200 cell/strain).
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pgen-1002732-g005: Mum2 and Slz1 interact with Ime4 and are required for m6A formation.A) Western analysis for co-immunoprecipitation of Mum2 (left panels) and Slz1 (right panels) with Ime4. HA-tagged Mum2 or Slz1 was immunoprecipitated from cellular extracts 3 hours after induction of meiosis and probed for interaction with myc-tagged Ime4 (SAy1232, SAy1253, respectively). A myc-Ime4 (SAy914) strain without HA-tags served as a control. Arrows in the IP lanes indicate IgG bands. B) Western analysis for HA-tagged Mum2 protein (SAy1235) or HA-tagged Slz1 protein (SAy1254) throughout meiosis; Pgk1 protein serves as loading control. C) Quantification of m6A on mRNA three hours after meiotic starvation, when m6A accumulation is maximal in wild-type cells (SAy821). Deleting any one of ime4Δ/Δ (SAy771), mum2Δ/Δ (SAy1196) and slz1Δ/Δ (SAy1206) results in a reduction in m6A levels. D) Wild-type (SAy821), ime4Δ/Δ (SAy771), mum2Δ/Δ (SAy1196) and slz1Δ/Δ (SAy1206) daughter cell morphology upon RTG3 (top panels). Arrows indicate primary buds. The same strains were photographed after growth on SLAD for 6 days (bottom panels). E) Axial ratio quantification of primary daughter cells upon RTG3 for strains in (D). (F) FACS analysis of DNA synthesis in strains from (D) 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. G) Kinetics for meiotic nuclear divisions as assayed by DNA staining by DAPI in the strains from (C) (n = 200 cells/strain/time point). H) Number of asci with one, two, three/four, or no spores in strains from (D) after 24 hours in SPO medium (n = 200 cell/strain).

Mentions: To identify regulators of Ime4, we conducted a two-hybrid screen using full-length Ime4 as bait [33]. The two most abundant hits isolated from this screen were MUM2 (MUddled Meiosis 2) and SLZ1 (Sporulation-specific Leucine Zipper 1) both of which have previously been implicated in meiotic progression [34], [35], [36]. Our screen identified 28 independent clones of MUM2 and 8 independent clones of SLZ1. All 28 clones spanned the 3′ region of MUM2 and all 8 clones spanned the 3′ region of SLZ1, suggesting that the respective carboxy-terminal regions of these proteins are sufficient for conferring interaction with Ime4 (Figure S5). In support of the physical interactions revealed by two-hybrid analysis, Ime4 efficiently co-immunoprecipitated with Mum2 and, to a lesser extent, Slz1 (Figure 5A). Like Ime4, Mum2 and Slz1 were induced during starvation in SPO as determined by Western blotting (Figure 5B).


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

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

Mum2 and Slz1 interact with Ime4 and are required for m6A formation.A) Western analysis for co-immunoprecipitation of Mum2 (left panels) and Slz1 (right panels) with Ime4. HA-tagged Mum2 or Slz1 was immunoprecipitated from cellular extracts 3 hours after induction of meiosis and probed for interaction with myc-tagged Ime4 (SAy1232, SAy1253, respectively). A myc-Ime4 (SAy914) strain without HA-tags served as a control. Arrows in the IP lanes indicate IgG bands. B) Western analysis for HA-tagged Mum2 protein (SAy1235) or HA-tagged Slz1 protein (SAy1254) throughout meiosis; Pgk1 protein serves as loading control. C) Quantification of m6A on mRNA three hours after meiotic starvation, when m6A accumulation is maximal in wild-type cells (SAy821). Deleting any one of ime4Δ/Δ (SAy771), mum2Δ/Δ (SAy1196) and slz1Δ/Δ (SAy1206) results in a reduction in m6A levels. D) Wild-type (SAy821), ime4Δ/Δ (SAy771), mum2Δ/Δ (SAy1196) and slz1Δ/Δ (SAy1206) daughter cell morphology upon RTG3 (top panels). Arrows indicate primary buds. The same strains were photographed after growth on SLAD for 6 days (bottom panels). E) Axial ratio quantification of primary daughter cells upon RTG3 for strains in (D). (F) FACS analysis of DNA synthesis in strains from (D) 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. G) Kinetics for meiotic nuclear divisions as assayed by DNA staining by DAPI in the strains from (C) (n = 200 cells/strain/time point). H) Number of asci with one, two, three/four, or no spores in strains from (D) after 24 hours in SPO medium (n = 200 cell/strain).
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Related In: Results  -  Collection

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pgen-1002732-g005: Mum2 and Slz1 interact with Ime4 and are required for m6A formation.A) Western analysis for co-immunoprecipitation of Mum2 (left panels) and Slz1 (right panels) with Ime4. HA-tagged Mum2 or Slz1 was immunoprecipitated from cellular extracts 3 hours after induction of meiosis and probed for interaction with myc-tagged Ime4 (SAy1232, SAy1253, respectively). A myc-Ime4 (SAy914) strain without HA-tags served as a control. Arrows in the IP lanes indicate IgG bands. B) Western analysis for HA-tagged Mum2 protein (SAy1235) or HA-tagged Slz1 protein (SAy1254) throughout meiosis; Pgk1 protein serves as loading control. C) Quantification of m6A on mRNA three hours after meiotic starvation, when m6A accumulation is maximal in wild-type cells (SAy821). Deleting any one of ime4Δ/Δ (SAy771), mum2Δ/Δ (SAy1196) and slz1Δ/Δ (SAy1206) results in a reduction in m6A levels. D) Wild-type (SAy821), ime4Δ/Δ (SAy771), mum2Δ/Δ (SAy1196) and slz1Δ/Δ (SAy1206) daughter cell morphology upon RTG3 (top panels). Arrows indicate primary buds. The same strains were photographed after growth on SLAD for 6 days (bottom panels). E) Axial ratio quantification of primary daughter cells upon RTG3 for strains in (D). (F) FACS analysis of DNA synthesis in strains from (D) 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. G) Kinetics for meiotic nuclear divisions as assayed by DNA staining by DAPI in the strains from (C) (n = 200 cells/strain/time point). H) Number of asci with one, two, three/four, or no spores in strains from (D) after 24 hours in SPO medium (n = 200 cell/strain).
Mentions: To identify regulators of Ime4, we conducted a two-hybrid screen using full-length Ime4 as bait [33]. The two most abundant hits isolated from this screen were MUM2 (MUddled Meiosis 2) and SLZ1 (Sporulation-specific Leucine Zipper 1) both of which have previously been implicated in meiotic progression [34], [35], [36]. Our screen identified 28 independent clones of MUM2 and 8 independent clones of SLZ1. All 28 clones spanned the 3′ region of MUM2 and all 8 clones spanned the 3′ region of SLZ1, suggesting that the respective carboxy-terminal regions of these proteins are sufficient for conferring interaction with Ime4 (Figure S5). In support of the physical interactions revealed by two-hybrid analysis, Ime4 efficiently co-immunoprecipitated with Mum2 and, to a lesser extent, Slz1 (Figure 5A). Like Ime4, Mum2 and Slz1 were induced during starvation in SPO as determined by Western blotting (Figure 5B).

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