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Transcription of the mating-type-regulated lncRNA IRT1 is governed by TORC1 and PKA

View Article: PubMed Central - PubMed

ABSTRACT

Cell fate decisions are controlled by multiple cell-intrinsic and -extrinsic factors. In budding yeast, the decision to enter gametogenesis or sporulation is dictated by nutrient availability and mating type. Recently, we showed that in diploid cells harbouring opposite mating types (MATa and MATα), the protein kinase A (PKA) and target of rapamycin complex I (TORC1) signalling pathways integrate at the promoter of the master regulatory transcription factor IME1 to control sporulation via nutrient availability (Weidberg, et al. 2016). In cells with a single mating type (MATa or MATα), however, IME1 is repressed by transcription through the IME1 promoter of a long non-coding RNA called IRT1, which prevents this cell type from undergoing sporulation. Here, we investigated the role of nutrient signalling in mating-type control of IME1. We find that expression of IRT1, like IME1 itself, depends on nutrient availability and the activities of PKA and TORC1. IRT1 transcription is repressed when nutrients are ample and TORC1 and PKA are active. In contrast, inhibition of PKA and TORC1 is sufficient to recruit Rme1 to the IRT1 promoter and induce IRT1-mediated repression of IME1. Finally, we provide evidence that IRT1 and IME1 are co-repressed by the Tup1–Cyc8 complex when nutrients are ample. Thus, in cells with a single mating-type nutrient availability regulates mating-type repression of IME1 and sporulation. Our results indicate that there is a hierarchy between nutrient and mating-type signals in controlling the decision to enter sporulation.

No MeSH data available.


Related in: MedlinePlus

IRT1 is induced in rich medium when PKA and TORC1 are inhibited. a Overview of IRT1/IME1 locus (top), and northern blot of IRT1 expression (bottom). Haploid cells harbouring the tpk1M164G, tpk2Δ, tpk3Δ alleles (tpk1as) (FW1760) were grown overnight in YPD, diluted into YPD plus 1NM-PP1 or rapamycin/1NM-PP1, and samples were taken at the indicated time points. Total RNA was isolated, separated by gel electrophoresis, blotted and probed for IRT1.bIRT1 RNA quantification in haploid cells harbouring tpk1as (FW1760). Cells were grown overnight in YPD, diluted into fresh YPD in absence or presence of rapamycin, 1NM-PP1, or rapamycin/1NM-PP1. Samples were taken at the indicated time points. Total RNA was isolated, reverse transcribed, and IRT1 mRNA levels were measured by quantitative PCR. Signals were normalized to ACT1 levels. The average signals of two biological experiments are shown. c Cells were grown and treated as in b. Rme1 binding to the IRT1 promoter was measured by chromatin immunoprecipitation at 4 h after treatment. Cells were fixed with formaldehyde, following immunoprecipitation of Rme1 tagged with 3xV5 epitope (FW1765) from chromatin extracts (see “Materials and methods” for details). The recovered DNA was quantified by real-time PCR with primers corresponding to the IRT1 promoter (pIRT1). Signals were normalized to the silent mating-type locus (HMR), which does not bind Rme1
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Fig1: IRT1 is induced in rich medium when PKA and TORC1 are inhibited. a Overview of IRT1/IME1 locus (top), and northern blot of IRT1 expression (bottom). Haploid cells harbouring the tpk1M164G, tpk2Δ, tpk3Δ alleles (tpk1as) (FW1760) were grown overnight in YPD, diluted into YPD plus 1NM-PP1 or rapamycin/1NM-PP1, and samples were taken at the indicated time points. Total RNA was isolated, separated by gel electrophoresis, blotted and probed for IRT1.bIRT1 RNA quantification in haploid cells harbouring tpk1as (FW1760). Cells were grown overnight in YPD, diluted into fresh YPD in absence or presence of rapamycin, 1NM-PP1, or rapamycin/1NM-PP1. Samples were taken at the indicated time points. Total RNA was isolated, reverse transcribed, and IRT1 mRNA levels were measured by quantitative PCR. Signals were normalized to ACT1 levels. The average signals of two biological experiments are shown. c Cells were grown and treated as in b. Rme1 binding to the IRT1 promoter was measured by chromatin immunoprecipitation at 4 h after treatment. Cells were fixed with formaldehyde, following immunoprecipitation of Rme1 tagged with 3xV5 epitope (FW1765) from chromatin extracts (see “Materials and methods” for details). The recovered DNA was quantified by real-time PCR with primers corresponding to the IRT1 promoter (pIRT1). Signals were normalized to the silent mating-type locus (HMR), which does not bind Rme1

Mentions: For Fig. 1, cells were grown overnight in YPD (1 % yeast extract, 2 % peptone, 2 % glucose) at 30 °C, then diluted to fresh YPD (OD600 = 1) and treated with different drugs. For Fig. 2, cells were grown overnight and diluted to OD600 = 2, and samples were taken at the indicated time points.Fig. 1


Transcription of the mating-type-regulated lncRNA IRT1 is governed by TORC1 and PKA
IRT1 is induced in rich medium when PKA and TORC1 are inhibited. a Overview of IRT1/IME1 locus (top), and northern blot of IRT1 expression (bottom). Haploid cells harbouring the tpk1M164G, tpk2Δ, tpk3Δ alleles (tpk1as) (FW1760) were grown overnight in YPD, diluted into YPD plus 1NM-PP1 or rapamycin/1NM-PP1, and samples were taken at the indicated time points. Total RNA was isolated, separated by gel electrophoresis, blotted and probed for IRT1.bIRT1 RNA quantification in haploid cells harbouring tpk1as (FW1760). Cells were grown overnight in YPD, diluted into fresh YPD in absence or presence of rapamycin, 1NM-PP1, or rapamycin/1NM-PP1. Samples were taken at the indicated time points. Total RNA was isolated, reverse transcribed, and IRT1 mRNA levels were measured by quantitative PCR. Signals were normalized to ACT1 levels. The average signals of two biological experiments are shown. c Cells were grown and treated as in b. Rme1 binding to the IRT1 promoter was measured by chromatin immunoprecipitation at 4 h after treatment. Cells were fixed with formaldehyde, following immunoprecipitation of Rme1 tagged with 3xV5 epitope (FW1765) from chromatin extracts (see “Materials and methods” for details). The recovered DNA was quantified by real-time PCR with primers corresponding to the IRT1 promoter (pIRT1). Signals were normalized to the silent mating-type locus (HMR), which does not bind Rme1
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5383673&req=5

Fig1: IRT1 is induced in rich medium when PKA and TORC1 are inhibited. a Overview of IRT1/IME1 locus (top), and northern blot of IRT1 expression (bottom). Haploid cells harbouring the tpk1M164G, tpk2Δ, tpk3Δ alleles (tpk1as) (FW1760) were grown overnight in YPD, diluted into YPD plus 1NM-PP1 or rapamycin/1NM-PP1, and samples were taken at the indicated time points. Total RNA was isolated, separated by gel electrophoresis, blotted and probed for IRT1.bIRT1 RNA quantification in haploid cells harbouring tpk1as (FW1760). Cells were grown overnight in YPD, diluted into fresh YPD in absence or presence of rapamycin, 1NM-PP1, or rapamycin/1NM-PP1. Samples were taken at the indicated time points. Total RNA was isolated, reverse transcribed, and IRT1 mRNA levels were measured by quantitative PCR. Signals were normalized to ACT1 levels. The average signals of two biological experiments are shown. c Cells were grown and treated as in b. Rme1 binding to the IRT1 promoter was measured by chromatin immunoprecipitation at 4 h after treatment. Cells were fixed with formaldehyde, following immunoprecipitation of Rme1 tagged with 3xV5 epitope (FW1765) from chromatin extracts (see “Materials and methods” for details). The recovered DNA was quantified by real-time PCR with primers corresponding to the IRT1 promoter (pIRT1). Signals were normalized to the silent mating-type locus (HMR), which does not bind Rme1
Mentions: For Fig. 1, cells were grown overnight in YPD (1 % yeast extract, 2 % peptone, 2 % glucose) at 30 °C, then diluted to fresh YPD (OD600 = 1) and treated with different drugs. For Fig. 2, cells were grown overnight and diluted to OD600 = 2, and samples were taken at the indicated time points.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Cell fate decisions are controlled by multiple cell-intrinsic and -extrinsic factors. In budding yeast, the decision to enter gametogenesis or sporulation is dictated by nutrient availability and mating type. Recently, we showed that in diploid cells harbouring opposite mating types (MATa and MATα), the protein kinase A (PKA) and target of rapamycin complex I (TORC1) signalling pathways integrate at the promoter of the master regulatory transcription factor IME1 to control sporulation via nutrient availability (Weidberg, et al. 2016). In cells with a single mating type (MATa or MATα), however, IME1 is repressed by transcription through the IME1 promoter of a long non-coding RNA called IRT1, which prevents this cell type from undergoing sporulation. Here, we investigated the role of nutrient signalling in mating-type control of IME1. We find that expression of IRT1, like IME1 itself, depends on nutrient availability and the activities of PKA and TORC1. IRT1 transcription is repressed when nutrients are ample and TORC1 and PKA are active. In contrast, inhibition of PKA and TORC1 is sufficient to recruit Rme1 to the IRT1 promoter and induce IRT1-mediated repression of IME1. Finally, we provide evidence that IRT1 and IME1 are co-repressed by the Tup1–Cyc8 complex when nutrients are ample. Thus, in cells with a single mating-type nutrient availability regulates mating-type repression of IME1 and sporulation. Our results indicate that there is a hierarchy between nutrient and mating-type signals in controlling the decision to enter sporulation.

No MeSH data available.


Related in: MedlinePlus