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Proteasomal degradation of Sfp1 contributes to the repression of ribosome biogenesis during starvation and is mediated by the proteasome activator Blm10.

Lopez AD, Tar K, Krügel U, Dange T, Ros IG, Schmidt M - Mol. Biol. Cell (2011)

Bottom Line: Repression of RP gene transcription appears to be regulated predominantly by posttranslational modification and cellular localization of transcriptional activators.We report here that one of these factors, Sfp1, is degraded by the proteasome and that the proteasome activator Blm10 is required for regulated Sfp1 degradation.Thus we conclude that proteasomal degradation of Sfp1 is mediated by Blm10 and contributes to the repression of ribosome biogenesis under nutrient depletion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
The regulation of ribosomal protein (RP) gene transcription is tightly linked to the nutrient status of the cell and is under the control of metabolic signaling pathways. In Saccharomyces cerevisiae several transcriptional activators mediate efficient RP gene transcription during logarithmic growth and dissociate from RP gene promoters upon nutrient limitation. Repression of RP gene transcription appears to be regulated predominantly by posttranslational modification and cellular localization of transcriptional activators. We report here that one of these factors, Sfp1, is degraded by the proteasome and that the proteasome activator Blm10 is required for regulated Sfp1 degradation. Loss of Blm10 results in the stabilization and increased nuclear abundance of Sfp1 during nutrient limitation, increased transcription of RP genes, increased levels of RPs, and decreased rapamycin-induced repression of RP genes. Thus we conclude that proteasomal degradation of Sfp1 is mediated by Blm10 and contributes to the repression of ribosome biogenesis under nutrient depletion.

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RP gene transcription is increased in BLM10-deleted cells after the diauxic shift. Expression of RP genes (RPS6A, RPL30, RPL3, RPL28, RPL11) was analyzed using qRT-PCR. Cycle threshold (CT) values were normalized to ACT1 expression levels. Data are reported as mean ± SEM. A single asterisk indicates a P-value < 0.05; a double asterisk indicates P < 0.01. (A) RP gene transcription is repressed in PDS. The level of RP gene expression in WT (yMS524) cells was determined in log and PDS phase in four independent experiments. PDS values are normalized to log expression levels. (B and C) Up-regulation of RP gene transcription in BLM10-deleted cells in PDS. RP gene transcription was analyzed in WT (yMS524) and blm10Δ (yMS63) in log (B) and PDS (C). RP gene expression in blm10Δ cells was normalized to WT expression in log or PDS phase. The values represent the mean of three independent experiments. (D) BLM10-deleted cells are less responsive to rapamycin-induced RP gene repression. mRNA expression of RP genes was analyzed 1 h after the addition of rapamycin (50 ng/ml) to logarithmically growing WT (yMS524) and BLM10-deleted (yMS63) cells in YPD. The values of RP gene expression levels after rapamycin treatment were normalized to the untreated control strains. The values represent the mean of three independent experiments.
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Figure 3: RP gene transcription is increased in BLM10-deleted cells after the diauxic shift. Expression of RP genes (RPS6A, RPL30, RPL3, RPL28, RPL11) was analyzed using qRT-PCR. Cycle threshold (CT) values were normalized to ACT1 expression levels. Data are reported as mean ± SEM. A single asterisk indicates a P-value < 0.05; a double asterisk indicates P < 0.01. (A) RP gene transcription is repressed in PDS. The level of RP gene expression in WT (yMS524) cells was determined in log and PDS phase in four independent experiments. PDS values are normalized to log expression levels. (B and C) Up-regulation of RP gene transcription in BLM10-deleted cells in PDS. RP gene transcription was analyzed in WT (yMS524) and blm10Δ (yMS63) in log (B) and PDS (C). RP gene expression in blm10Δ cells was normalized to WT expression in log or PDS phase. The values represent the mean of three independent experiments. (D) BLM10-deleted cells are less responsive to rapamycin-induced RP gene repression. mRNA expression of RP genes was analyzed 1 h after the addition of rapamycin (50 ng/ml) to logarithmically growing WT (yMS524) and BLM10-deleted (yMS63) cells in YPD. The values of RP gene expression levels after rapamycin treatment were normalized to the untreated control strains. The values represent the mean of three independent experiments.

Mentions: An alternative explanation for elevated RP levels in BLM10-deleted cells would be dysregulation of RP gene transcription. We therefore determined mRNA levels of five RP genes by quantitative real-time PCR (qRT-PCR) in WT and blm10Δ cells in the different growth phases. RP gene transcription is strongly repressed after the diauxic shift in WT cells (Figure 3A; Brauer et al., 2005). Loss of BLM10 did not affect RP gene transcription in log phase (Figure 3B), in agreement with unchanged RP levels observed under the same conditions (Figure 2B). After the diauxic shift, however, RP gene transcription was elevated in the absence of BLM10 (Figure 3C).


Proteasomal degradation of Sfp1 contributes to the repression of ribosome biogenesis during starvation and is mediated by the proteasome activator Blm10.

Lopez AD, Tar K, Krügel U, Dange T, Ros IG, Schmidt M - Mol. Biol. Cell (2011)

RP gene transcription is increased in BLM10-deleted cells after the diauxic shift. Expression of RP genes (RPS6A, RPL30, RPL3, RPL28, RPL11) was analyzed using qRT-PCR. Cycle threshold (CT) values were normalized to ACT1 expression levels. Data are reported as mean ± SEM. A single asterisk indicates a P-value < 0.05; a double asterisk indicates P < 0.01. (A) RP gene transcription is repressed in PDS. The level of RP gene expression in WT (yMS524) cells was determined in log and PDS phase in four independent experiments. PDS values are normalized to log expression levels. (B and C) Up-regulation of RP gene transcription in BLM10-deleted cells in PDS. RP gene transcription was analyzed in WT (yMS524) and blm10Δ (yMS63) in log (B) and PDS (C). RP gene expression in blm10Δ cells was normalized to WT expression in log or PDS phase. The values represent the mean of three independent experiments. (D) BLM10-deleted cells are less responsive to rapamycin-induced RP gene repression. mRNA expression of RP genes was analyzed 1 h after the addition of rapamycin (50 ng/ml) to logarithmically growing WT (yMS524) and BLM10-deleted (yMS63) cells in YPD. The values of RP gene expression levels after rapamycin treatment were normalized to the untreated control strains. The values represent the mean of three independent experiments.
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Related In: Results  -  Collection

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Figure 3: RP gene transcription is increased in BLM10-deleted cells after the diauxic shift. Expression of RP genes (RPS6A, RPL30, RPL3, RPL28, RPL11) was analyzed using qRT-PCR. Cycle threshold (CT) values were normalized to ACT1 expression levels. Data are reported as mean ± SEM. A single asterisk indicates a P-value < 0.05; a double asterisk indicates P < 0.01. (A) RP gene transcription is repressed in PDS. The level of RP gene expression in WT (yMS524) cells was determined in log and PDS phase in four independent experiments. PDS values are normalized to log expression levels. (B and C) Up-regulation of RP gene transcription in BLM10-deleted cells in PDS. RP gene transcription was analyzed in WT (yMS524) and blm10Δ (yMS63) in log (B) and PDS (C). RP gene expression in blm10Δ cells was normalized to WT expression in log or PDS phase. The values represent the mean of three independent experiments. (D) BLM10-deleted cells are less responsive to rapamycin-induced RP gene repression. mRNA expression of RP genes was analyzed 1 h after the addition of rapamycin (50 ng/ml) to logarithmically growing WT (yMS524) and BLM10-deleted (yMS63) cells in YPD. The values of RP gene expression levels after rapamycin treatment were normalized to the untreated control strains. The values represent the mean of three independent experiments.
Mentions: An alternative explanation for elevated RP levels in BLM10-deleted cells would be dysregulation of RP gene transcription. We therefore determined mRNA levels of five RP genes by quantitative real-time PCR (qRT-PCR) in WT and blm10Δ cells in the different growth phases. RP gene transcription is strongly repressed after the diauxic shift in WT cells (Figure 3A; Brauer et al., 2005). Loss of BLM10 did not affect RP gene transcription in log phase (Figure 3B), in agreement with unchanged RP levels observed under the same conditions (Figure 2B). After the diauxic shift, however, RP gene transcription was elevated in the absence of BLM10 (Figure 3C).

Bottom Line: Repression of RP gene transcription appears to be regulated predominantly by posttranslational modification and cellular localization of transcriptional activators.We report here that one of these factors, Sfp1, is degraded by the proteasome and that the proteasome activator Blm10 is required for regulated Sfp1 degradation.Thus we conclude that proteasomal degradation of Sfp1 is mediated by Blm10 and contributes to the repression of ribosome biogenesis under nutrient depletion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
The regulation of ribosomal protein (RP) gene transcription is tightly linked to the nutrient status of the cell and is under the control of metabolic signaling pathways. In Saccharomyces cerevisiae several transcriptional activators mediate efficient RP gene transcription during logarithmic growth and dissociate from RP gene promoters upon nutrient limitation. Repression of RP gene transcription appears to be regulated predominantly by posttranslational modification and cellular localization of transcriptional activators. We report here that one of these factors, Sfp1, is degraded by the proteasome and that the proteasome activator Blm10 is required for regulated Sfp1 degradation. Loss of Blm10 results in the stabilization and increased nuclear abundance of Sfp1 during nutrient limitation, increased transcription of RP genes, increased levels of RPs, and decreased rapamycin-induced repression of RP genes. Thus we conclude that proteasomal degradation of Sfp1 is mediated by Blm10 and contributes to the repression of ribosome biogenesis under nutrient depletion.

Show MeSH
Related in: MedlinePlus