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Differential Phosphorylation of RNA Polymerase III and the Initiation Factor TFIIIB in Saccharomyces cerevisiae.

Lee J, Moir RD, Willis IM - PLoS ONE (2015)

Bottom Line: However, numerous lines of evidence suggest greater complexity in the regulatory network including the phosphoregulation of other pol III components.A relatively high stoichiometry of phosphorylation was observed for several of these proteins and the Rpc82 subunit of the polymerase and the Bdp1 subunit of TFIIIB were found to be differentially phosphorylated.Alanine substitutions at the four phosphosites cause hyper-repression of transcription indicating that phosphorylation of Bdp1 opposes Maf1-mediated repression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
The production of ribosomes and tRNAs for protein synthesis has a high energetic cost and is under tight transcriptional control to ensure that the level of RNA synthesis is balanced with nutrient availability and the prevailing environmental conditions. In the RNA polymerase (pol) III system in yeast, nutrients and stress affect transcription through a bifurcated signaling pathway in which protein kinase A (PKA) and TORC1 activity directly or indirectly, through downstream kinases, alter the phosphorylation state and function of the Maf1 repressor and Rpc53, a TFIIF-like subunit of the polymerase. However, numerous lines of evidence suggest greater complexity in the regulatory network including the phosphoregulation of other pol III components. To address this issue, we systematically examined all 17 subunits of pol III along with the three subunits of the initiation factor TFIIIB for evidence of differential phosphorylation in response to inhibition of TORC1. A relatively high stoichiometry of phosphorylation was observed for several of these proteins and the Rpc82 subunit of the polymerase and the Bdp1 subunit of TFIIIB were found to be differentially phosphorylated. Bdp1 is phosphorylated on four major sites during exponential growth and the protein is variably dephosphorylated under conditions that inhibit tRNA gene transcription. PKA, the TORC1-regulated kinase Sch9 and protein kinase CK2 are all implicated in the phosphorylation of Bdp1. Alanine substitutions at the four phosphosites cause hyper-repression of transcription indicating that phosphorylation of Bdp1 opposes Maf1-mediated repression. The new findings suggest an integrated regulatory model for signaling events controlling pol III transcription.

No MeSH data available.


Related in: MedlinePlus

PKA and Sch9 regulate pol III transcription through additional effectors other than Maf1.Northern analysis of pol III transcription in analog-sensitive strains. pka-as, sch9-as and pka-as sch9-as strains (abbreviated to 4-as in panel B) were treated with 1NM-PP1 or drug vehicle for 1 hour. A. Pol III transcription reported by the amount of pre-tRNALeu transcript is normalized to U3 snRNA, expressed relative to the untreated wild-type strain and indicated below each lane. Transcription is sensitive to loss of either PKA or Sch9 catalytic activity and the reduction is Maf1-dependent (right-most 2 lanes). Transcription for maf1Δ pka-as sch9-as normalized to its maf1Δ control. B. Pol III transcription is sensitive to loss of PKA and Sch9 activity in a strain that contains alanine-substituted Maf1 (MAF1-7SA). An ATP analog-insensitive maf1Δ::natR strain and a pka-as sch9-as maf1Δ::natR strain, each containing a plasmid with MAF1 wild-type (WT) or 7SA alleles were assayed and quantified as in panel A using U4 snRNA for normalization. The framed regions in panels A and B represent non-adjacent lanes from the same gels.
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pone.0127225.g001: PKA and Sch9 regulate pol III transcription through additional effectors other than Maf1.Northern analysis of pol III transcription in analog-sensitive strains. pka-as, sch9-as and pka-as sch9-as strains (abbreviated to 4-as in panel B) were treated with 1NM-PP1 or drug vehicle for 1 hour. A. Pol III transcription reported by the amount of pre-tRNALeu transcript is normalized to U3 snRNA, expressed relative to the untreated wild-type strain and indicated below each lane. Transcription is sensitive to loss of either PKA or Sch9 catalytic activity and the reduction is Maf1-dependent (right-most 2 lanes). Transcription for maf1Δ pka-as sch9-as normalized to its maf1Δ control. B. Pol III transcription is sensitive to loss of PKA and Sch9 activity in a strain that contains alanine-substituted Maf1 (MAF1-7SA). An ATP analog-insensitive maf1Δ::natR strain and a pka-as sch9-as maf1Δ::natR strain, each containing a plasmid with MAF1 wild-type (WT) or 7SA alleles were assayed and quantified as in panel A using U4 snRNA for normalization. The framed regions in panels A and B represent non-adjacent lanes from the same gels.

Mentions: We and others have shown that PKA and Sch9 redundantly phosphorylate yeast Maf1 at six sites (serines 90, 101, 177, 178, 209, 210) with a seventh site (serine 179) targeted specifically by Sch9 [5, 34, 35, 40–42]. Thus, the near complete loss of Maf1 phosphorylation at these sites in vivo under optimal growth conditions requires the simultaneous inhibition of both kinases. This was demonstrated by comparing the phosphorylation state of Maf1 in a series of ATP analog-sensitive strains (pka-as, sch9-as and pka/sch9-as) following the addition of 1NM-PP1 [34]. However, parallel experiments to examine the inhibitory effects of the ATP analog on pol III transcription in these strains have not previously been reported (see Fig 1). Interestingly, despite the ability of PKA and Sch9 to buffer one another with respect to Maf1 phosphorylation [34], 1NM-PP1 inhibition of either kinase caused a significant (>50%) reduction in pol III transcription as determined by pre-tRNALeu northern analysis (Fig 1A). In this assay, the level of short-lived pre-tRNALeu molecules reports global tRNA synthesis and therefore pol III transcription [10, 43]. Unlike the pka-as strain, the sch9-as strain showed a modest decrease in pre-tRNALeu synthesis in the absence of 1NM-PP1 with analog addition causing a substantial further decrease. This effect was amplified in the pka/sch9-as strain (Fig 1A). Notably, reduced transcription in either the absence or the presence of 1NM-PP1 was fully dependent on Maf1 since the changes were quantitatively blocked in the pka/sch9-as maf1∆ strain (Fig 1A, right hand lanes). In addition, we observed that the effect of 1NM-PP1 treatment on each of the three analog-sensitive strains (>50% reduction in pre-tRNALeu synthesis after 1 hour) was significantly greater than the effect of the Maf1 7SA mutant under optimal growth conditions (25% reduction or 0.75 ± 0.07, n = 8, SEM, relative to 1.0 for wild-type, in pre-tRNALeu synthesis with mutation of all seven PKA and Sch9 sites to alanine) [44]. This suggested that PKA and Sch9 may have other targets in the pol III machinery that impact the level of transcription. To test this possibility, we examined the effect of 1NM-PP1 on pre-tRNALeu synthesis in wild-type and pka/sch9-as strains in the presence or absence of Maf1 7SA. If control of pol III transcription by PKA and Sch9 is exercised exclusively through Maf1, 1NM-PP1 should have no effect in the pka/sch9-as MAF1-7SA strain. This is not the case (Fig 1B); pre-tRNALeu synthesis in the pka/sch9-as MAF1-7SA was significantly reduced (comparable to wild-type Maf1) upon addition of the analog. Thus, these data support the existence of one or more additional substrates for PKA and Sch9 in the pol III system and suggest that the putative substrates are phosphorylated under normal growth conditions and that their dephosphorylation promotes Maf1-dependent repression.


Differential Phosphorylation of RNA Polymerase III and the Initiation Factor TFIIIB in Saccharomyces cerevisiae.

Lee J, Moir RD, Willis IM - PLoS ONE (2015)

PKA and Sch9 regulate pol III transcription through additional effectors other than Maf1.Northern analysis of pol III transcription in analog-sensitive strains. pka-as, sch9-as and pka-as sch9-as strains (abbreviated to 4-as in panel B) were treated with 1NM-PP1 or drug vehicle for 1 hour. A. Pol III transcription reported by the amount of pre-tRNALeu transcript is normalized to U3 snRNA, expressed relative to the untreated wild-type strain and indicated below each lane. Transcription is sensitive to loss of either PKA or Sch9 catalytic activity and the reduction is Maf1-dependent (right-most 2 lanes). Transcription for maf1Δ pka-as sch9-as normalized to its maf1Δ control. B. Pol III transcription is sensitive to loss of PKA and Sch9 activity in a strain that contains alanine-substituted Maf1 (MAF1-7SA). An ATP analog-insensitive maf1Δ::natR strain and a pka-as sch9-as maf1Δ::natR strain, each containing a plasmid with MAF1 wild-type (WT) or 7SA alleles were assayed and quantified as in panel A using U4 snRNA for normalization. The framed regions in panels A and B represent non-adjacent lanes from the same gels.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone.0127225.g001: PKA and Sch9 regulate pol III transcription through additional effectors other than Maf1.Northern analysis of pol III transcription in analog-sensitive strains. pka-as, sch9-as and pka-as sch9-as strains (abbreviated to 4-as in panel B) were treated with 1NM-PP1 or drug vehicle for 1 hour. A. Pol III transcription reported by the amount of pre-tRNALeu transcript is normalized to U3 snRNA, expressed relative to the untreated wild-type strain and indicated below each lane. Transcription is sensitive to loss of either PKA or Sch9 catalytic activity and the reduction is Maf1-dependent (right-most 2 lanes). Transcription for maf1Δ pka-as sch9-as normalized to its maf1Δ control. B. Pol III transcription is sensitive to loss of PKA and Sch9 activity in a strain that contains alanine-substituted Maf1 (MAF1-7SA). An ATP analog-insensitive maf1Δ::natR strain and a pka-as sch9-as maf1Δ::natR strain, each containing a plasmid with MAF1 wild-type (WT) or 7SA alleles were assayed and quantified as in panel A using U4 snRNA for normalization. The framed regions in panels A and B represent non-adjacent lanes from the same gels.
Mentions: We and others have shown that PKA and Sch9 redundantly phosphorylate yeast Maf1 at six sites (serines 90, 101, 177, 178, 209, 210) with a seventh site (serine 179) targeted specifically by Sch9 [5, 34, 35, 40–42]. Thus, the near complete loss of Maf1 phosphorylation at these sites in vivo under optimal growth conditions requires the simultaneous inhibition of both kinases. This was demonstrated by comparing the phosphorylation state of Maf1 in a series of ATP analog-sensitive strains (pka-as, sch9-as and pka/sch9-as) following the addition of 1NM-PP1 [34]. However, parallel experiments to examine the inhibitory effects of the ATP analog on pol III transcription in these strains have not previously been reported (see Fig 1). Interestingly, despite the ability of PKA and Sch9 to buffer one another with respect to Maf1 phosphorylation [34], 1NM-PP1 inhibition of either kinase caused a significant (>50%) reduction in pol III transcription as determined by pre-tRNALeu northern analysis (Fig 1A). In this assay, the level of short-lived pre-tRNALeu molecules reports global tRNA synthesis and therefore pol III transcription [10, 43]. Unlike the pka-as strain, the sch9-as strain showed a modest decrease in pre-tRNALeu synthesis in the absence of 1NM-PP1 with analog addition causing a substantial further decrease. This effect was amplified in the pka/sch9-as strain (Fig 1A). Notably, reduced transcription in either the absence or the presence of 1NM-PP1 was fully dependent on Maf1 since the changes were quantitatively blocked in the pka/sch9-as maf1∆ strain (Fig 1A, right hand lanes). In addition, we observed that the effect of 1NM-PP1 treatment on each of the three analog-sensitive strains (>50% reduction in pre-tRNALeu synthesis after 1 hour) was significantly greater than the effect of the Maf1 7SA mutant under optimal growth conditions (25% reduction or 0.75 ± 0.07, n = 8, SEM, relative to 1.0 for wild-type, in pre-tRNALeu synthesis with mutation of all seven PKA and Sch9 sites to alanine) [44]. This suggested that PKA and Sch9 may have other targets in the pol III machinery that impact the level of transcription. To test this possibility, we examined the effect of 1NM-PP1 on pre-tRNALeu synthesis in wild-type and pka/sch9-as strains in the presence or absence of Maf1 7SA. If control of pol III transcription by PKA and Sch9 is exercised exclusively through Maf1, 1NM-PP1 should have no effect in the pka/sch9-as MAF1-7SA strain. This is not the case (Fig 1B); pre-tRNALeu synthesis in the pka/sch9-as MAF1-7SA was significantly reduced (comparable to wild-type Maf1) upon addition of the analog. Thus, these data support the existence of one or more additional substrates for PKA and Sch9 in the pol III system and suggest that the putative substrates are phosphorylated under normal growth conditions and that their dephosphorylation promotes Maf1-dependent repression.

Bottom Line: However, numerous lines of evidence suggest greater complexity in the regulatory network including the phosphoregulation of other pol III components.A relatively high stoichiometry of phosphorylation was observed for several of these proteins and the Rpc82 subunit of the polymerase and the Bdp1 subunit of TFIIIB were found to be differentially phosphorylated.Alanine substitutions at the four phosphosites cause hyper-repression of transcription indicating that phosphorylation of Bdp1 opposes Maf1-mediated repression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
The production of ribosomes and tRNAs for protein synthesis has a high energetic cost and is under tight transcriptional control to ensure that the level of RNA synthesis is balanced with nutrient availability and the prevailing environmental conditions. In the RNA polymerase (pol) III system in yeast, nutrients and stress affect transcription through a bifurcated signaling pathway in which protein kinase A (PKA) and TORC1 activity directly or indirectly, through downstream kinases, alter the phosphorylation state and function of the Maf1 repressor and Rpc53, a TFIIF-like subunit of the polymerase. However, numerous lines of evidence suggest greater complexity in the regulatory network including the phosphoregulation of other pol III components. To address this issue, we systematically examined all 17 subunits of pol III along with the three subunits of the initiation factor TFIIIB for evidence of differential phosphorylation in response to inhibition of TORC1. A relatively high stoichiometry of phosphorylation was observed for several of these proteins and the Rpc82 subunit of the polymerase and the Bdp1 subunit of TFIIIB were found to be differentially phosphorylated. Bdp1 is phosphorylated on four major sites during exponential growth and the protein is variably dephosphorylated under conditions that inhibit tRNA gene transcription. PKA, the TORC1-regulated kinase Sch9 and protein kinase CK2 are all implicated in the phosphorylation of Bdp1. Alanine substitutions at the four phosphosites cause hyper-repression of transcription indicating that phosphorylation of Bdp1 opposes Maf1-mediated repression. The new findings suggest an integrated regulatory model for signaling events controlling pol III transcription.

No MeSH data available.


Related in: MedlinePlus