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Acetylation of drosha on the N-terminus inhibits its degradation by ubiquitination.

Tang X, Wen S, Zheng D, Tucker L, Cao L, Pantazatos D, Moss SF, Ramratnam B - PLoS ONE (2013)

Bottom Line: TSA increases miRNA-143 production in a miRNA sensor assay and in a qPCR analysis in HEK293T cells.Furthermore, the N-terminal, but not the C-terminal Drosha can be acetylated by multiple acetyl transferases including p300, CBP and GCN5.Our findings establish a central mechanism of protein homeostasis as playing a critical role in miRNA biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Retrovirology, Division of Infectious Diseases, Department of Medicine, Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA.

ABSTRACT
The RNase III enzyme Drosha initiates microRNA (miRNA) biogenesis in the nucleus by cleaving primary miRNA transcripts into shorter precursor molecules that are subsequently exported into the cytoplasm for further processing. While numerous disease states appear to be associated with aberrant expression of Drosha, the molecular mechanisms that regulate its protein levels are largely unknown. Here, we report that ubiquitination and acetylation regulate Drosha protein levels oppositely. Deacetylase inhibitors trichostatin A (TSA) and nicotinamide (NIA) increase Drosha protein level as measured by western blot but have no effects on its mRNA level in HEK293T cells. TSA increases miRNA-143 production in a miRNA sensor assay and in a qPCR analysis in HEK293T cells. Treatment of AGS and HEK293T cells with proteasome inhibitors MG132 or Omuralide increases Drosha protein levels. Furthermore, the N-terminal, but not the C-terminal Drosha can be acetylated by multiple acetyl transferases including p300, CBP and GCN5. Acetylation of Drosha competes with its ubquitination, inhibiting the degradation induced by the ubiquitin-proteasome pathway, thereby increasing Drosha protein levels. Infection of the gastric mucosa AGS cells by H. pylori, the gastric cancer associated carcinogen, leads to the ubiquitination and reduction of Drosha protein levels. H. pylori infection of AGS cells has no significant effects on Drosha mRNA levels. Our findings establish a central mechanism of protein homeostasis as playing a critical role in miRNA biogenesis.

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Acetylation of Drosha increases its protein level.All experiments were repeated three times with similar results (p<0.05 by Student’s t-test). (A), Inhibition of deacetylases increases Drosha protein level measured by western blot. HEK293T cells were treated with vehicle, trichostatin A (TSA, 2 µM), or nicotinamide (NIA, 1 mM) overnight prior to harvest. (B) TSA, an HDAC inhibitor, increases ectopically expressed Drosha levels in HEK293T upon transfection with a GFP-Drosha construct. (C) Inhibition of deacetylases have no effects on Drosha mRNA level measured by RT-PCR. (D)Inhibition of deacetylases increases Drosha acetylation. HEK293T cells were transfected with empty vector (EV) or GFP-Drosha. Twenty-four hours post-transfection, the cells were treated with TSA (2 µM) or NIA (1 mM) overnight. Whole cell lysates were prepared and immunoprecipitated with GFP antibody conjugated sepharose beads. The immunoprecipitates were resolved and blotted with mouse monoclonal acetylated lysine antibody to detect Drosha acetylation. The same membrane was then reblotted to check Drosha protein level. (E) Multiple acetyl transferases acetylate Drosha. A GFP-Drosha construct was cotransfected with an empty vector, p300, CBP, PCAF or GCN5 construct respectively into HEK293T cells. Forty-eight hours post-transfection, half of the cells was used to extract total RNA for checking the mRNA levels of GFP-Drosha. Another half of the cells was used to prepare whole cell lysates for detecting Drosha acetylation as in Figure1C.
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pone-0072503-g001: Acetylation of Drosha increases its protein level.All experiments were repeated three times with similar results (p<0.05 by Student’s t-test). (A), Inhibition of deacetylases increases Drosha protein level measured by western blot. HEK293T cells were treated with vehicle, trichostatin A (TSA, 2 µM), or nicotinamide (NIA, 1 mM) overnight prior to harvest. (B) TSA, an HDAC inhibitor, increases ectopically expressed Drosha levels in HEK293T upon transfection with a GFP-Drosha construct. (C) Inhibition of deacetylases have no effects on Drosha mRNA level measured by RT-PCR. (D)Inhibition of deacetylases increases Drosha acetylation. HEK293T cells were transfected with empty vector (EV) or GFP-Drosha. Twenty-four hours post-transfection, the cells were treated with TSA (2 µM) or NIA (1 mM) overnight. Whole cell lysates were prepared and immunoprecipitated with GFP antibody conjugated sepharose beads. The immunoprecipitates were resolved and blotted with mouse monoclonal acetylated lysine antibody to detect Drosha acetylation. The same membrane was then reblotted to check Drosha protein level. (E) Multiple acetyl transferases acetylate Drosha. A GFP-Drosha construct was cotransfected with an empty vector, p300, CBP, PCAF or GCN5 construct respectively into HEK293T cells. Forty-eight hours post-transfection, half of the cells was used to extract total RNA for checking the mRNA levels of GFP-Drosha. Another half of the cells was used to prepare whole cell lysates for detecting Drosha acetylation as in Figure1C.

Mentions: Lysine residues serve as substrates for acetylation leading to overall stabilization of proteins [22]–[25]. To determine whether a similar dynamic existed for Drosha, we quantified protein levels in HEK293T cells after inhibition of deacetylases with trichostatin A (TSA) or nicotinamide (NIA). As seen in Figure 1A, inhibition of deacetylation lead to 3-fold increased endogenous Drosha protein levels indicating that acetylation was indeed involved in the regulation of Drosha protein stability. TSA had similar effect on exogenously expressed GFP-Drosha protein level (Figure 1B). Deacetylase inhibitors had no effects on Drosha mRNA levels (Figure 1C). We then examined the acetylation status of Drosha by western blotting and found that Drosha was constitutively acetylated and treatment with TSA or NIA further enhanced its acetylation (Figure 1D). To determine which acetyl transferase(s) were involved, we cotransfected GFP-Drosha with empty vector (EV), p300, CBP, PCAF or GCN5, respectively. The acetylation status and protein level of Drosha were determined by Western blot 48 hr posttransfection. In the meantime, GFP-Drosha mRNA levels were also measured by RT-PCR to rule out the effects of transfection efficiency on protein expression. We found that Drosha could be acetylated by multiple acetyl transferases including p300, CBP and GCN5 (Figure 1E).


Acetylation of drosha on the N-terminus inhibits its degradation by ubiquitination.

Tang X, Wen S, Zheng D, Tucker L, Cao L, Pantazatos D, Moss SF, Ramratnam B - PLoS ONE (2013)

Acetylation of Drosha increases its protein level.All experiments were repeated three times with similar results (p<0.05 by Student’s t-test). (A), Inhibition of deacetylases increases Drosha protein level measured by western blot. HEK293T cells were treated with vehicle, trichostatin A (TSA, 2 µM), or nicotinamide (NIA, 1 mM) overnight prior to harvest. (B) TSA, an HDAC inhibitor, increases ectopically expressed Drosha levels in HEK293T upon transfection with a GFP-Drosha construct. (C) Inhibition of deacetylases have no effects on Drosha mRNA level measured by RT-PCR. (D)Inhibition of deacetylases increases Drosha acetylation. HEK293T cells were transfected with empty vector (EV) or GFP-Drosha. Twenty-four hours post-transfection, the cells were treated with TSA (2 µM) or NIA (1 mM) overnight. Whole cell lysates were prepared and immunoprecipitated with GFP antibody conjugated sepharose beads. The immunoprecipitates were resolved and blotted with mouse monoclonal acetylated lysine antibody to detect Drosha acetylation. The same membrane was then reblotted to check Drosha protein level. (E) Multiple acetyl transferases acetylate Drosha. A GFP-Drosha construct was cotransfected with an empty vector, p300, CBP, PCAF or GCN5 construct respectively into HEK293T cells. Forty-eight hours post-transfection, half of the cells was used to extract total RNA for checking the mRNA levels of GFP-Drosha. Another half of the cells was used to prepare whole cell lysates for detecting Drosha acetylation as in Figure1C.
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getmorefigures.php?uid=PMC3757024&req=5

pone-0072503-g001: Acetylation of Drosha increases its protein level.All experiments were repeated three times with similar results (p<0.05 by Student’s t-test). (A), Inhibition of deacetylases increases Drosha protein level measured by western blot. HEK293T cells were treated with vehicle, trichostatin A (TSA, 2 µM), or nicotinamide (NIA, 1 mM) overnight prior to harvest. (B) TSA, an HDAC inhibitor, increases ectopically expressed Drosha levels in HEK293T upon transfection with a GFP-Drosha construct. (C) Inhibition of deacetylases have no effects on Drosha mRNA level measured by RT-PCR. (D)Inhibition of deacetylases increases Drosha acetylation. HEK293T cells were transfected with empty vector (EV) or GFP-Drosha. Twenty-four hours post-transfection, the cells were treated with TSA (2 µM) or NIA (1 mM) overnight. Whole cell lysates were prepared and immunoprecipitated with GFP antibody conjugated sepharose beads. The immunoprecipitates were resolved and blotted with mouse monoclonal acetylated lysine antibody to detect Drosha acetylation. The same membrane was then reblotted to check Drosha protein level. (E) Multiple acetyl transferases acetylate Drosha. A GFP-Drosha construct was cotransfected with an empty vector, p300, CBP, PCAF or GCN5 construct respectively into HEK293T cells. Forty-eight hours post-transfection, half of the cells was used to extract total RNA for checking the mRNA levels of GFP-Drosha. Another half of the cells was used to prepare whole cell lysates for detecting Drosha acetylation as in Figure1C.
Mentions: Lysine residues serve as substrates for acetylation leading to overall stabilization of proteins [22]–[25]. To determine whether a similar dynamic existed for Drosha, we quantified protein levels in HEK293T cells after inhibition of deacetylases with trichostatin A (TSA) or nicotinamide (NIA). As seen in Figure 1A, inhibition of deacetylation lead to 3-fold increased endogenous Drosha protein levels indicating that acetylation was indeed involved in the regulation of Drosha protein stability. TSA had similar effect on exogenously expressed GFP-Drosha protein level (Figure 1B). Deacetylase inhibitors had no effects on Drosha mRNA levels (Figure 1C). We then examined the acetylation status of Drosha by western blotting and found that Drosha was constitutively acetylated and treatment with TSA or NIA further enhanced its acetylation (Figure 1D). To determine which acetyl transferase(s) were involved, we cotransfected GFP-Drosha with empty vector (EV), p300, CBP, PCAF or GCN5, respectively. The acetylation status and protein level of Drosha were determined by Western blot 48 hr posttransfection. In the meantime, GFP-Drosha mRNA levels were also measured by RT-PCR to rule out the effects of transfection efficiency on protein expression. We found that Drosha could be acetylated by multiple acetyl transferases including p300, CBP and GCN5 (Figure 1E).

Bottom Line: TSA increases miRNA-143 production in a miRNA sensor assay and in a qPCR analysis in HEK293T cells.Furthermore, the N-terminal, but not the C-terminal Drosha can be acetylated by multiple acetyl transferases including p300, CBP and GCN5.Our findings establish a central mechanism of protein homeostasis as playing a critical role in miRNA biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Retrovirology, Division of Infectious Diseases, Department of Medicine, Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA.

ABSTRACT
The RNase III enzyme Drosha initiates microRNA (miRNA) biogenesis in the nucleus by cleaving primary miRNA transcripts into shorter precursor molecules that are subsequently exported into the cytoplasm for further processing. While numerous disease states appear to be associated with aberrant expression of Drosha, the molecular mechanisms that regulate its protein levels are largely unknown. Here, we report that ubiquitination and acetylation regulate Drosha protein levels oppositely. Deacetylase inhibitors trichostatin A (TSA) and nicotinamide (NIA) increase Drosha protein level as measured by western blot but have no effects on its mRNA level in HEK293T cells. TSA increases miRNA-143 production in a miRNA sensor assay and in a qPCR analysis in HEK293T cells. Treatment of AGS and HEK293T cells with proteasome inhibitors MG132 or Omuralide increases Drosha protein levels. Furthermore, the N-terminal, but not the C-terminal Drosha can be acetylated by multiple acetyl transferases including p300, CBP and GCN5. Acetylation of Drosha competes with its ubquitination, inhibiting the degradation induced by the ubiquitin-proteasome pathway, thereby increasing Drosha protein levels. Infection of the gastric mucosa AGS cells by H. pylori, the gastric cancer associated carcinogen, leads to the ubiquitination and reduction of Drosha protein levels. H. pylori infection of AGS cells has no significant effects on Drosha mRNA levels. Our findings establish a central mechanism of protein homeostasis as playing a critical role in miRNA biogenesis.

Show MeSH
Related in: MedlinePlus