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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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A constitutively active ubiquitin-proteasome pathway degrades Drosha.Whole cell lysates of the indicated cell types were prepared with IP Lysis Buffer (Pierce); 20 µg protein aliquots were used for western blots. All experiments were repeated three times with similar results (p<0.05 by Student’s t-test). (A) Abundant polyubiquitination was observed in multiple cell types using lysine 48-linkage specific polyubiquitin antibody. (B) Drosha is ubiquitinated in various cell types including HEK293T, HeLa and AGS. Left panel: HEK293T lysates were immunoprecipitated with control IgG or Rabbit Polyclonal Antibody to Drosha respectively. Right panel: Cell lysates were immunoprecipitated respectively with Rabbit Polyclonal Antibody to Drosha. The immunoprecipitates were resolved by SDS-PAGE and blotted with lysine 48-linkage specific polyubiquitin antibody. The same membrane was reblotted with Drosha Rabbit mAb. (C) Inhibition of the ubiquitin-proteasome pathway with MG132 (10 µM) increases endogenous Drosha protein level in AGS cells. GAPDH was used as a loading control. (D) Proteasomal inhibition increases exogenous GFP-Drosha expression level. HEK293T cells were transfected with GFP-Drosha. Twenty-four hours post-transfection, the cells were treated with 1 µM Clasto-Lactacystin-β-lactone (Omuralide) overnight. GFP-Drosha protein level was increased 3-fold following treatment.
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pone-0072503-g003: A constitutively active ubiquitin-proteasome pathway degrades Drosha.Whole cell lysates of the indicated cell types were prepared with IP Lysis Buffer (Pierce); 20 µg protein aliquots were used for western blots. All experiments were repeated three times with similar results (p<0.05 by Student’s t-test). (A) Abundant polyubiquitination was observed in multiple cell types using lysine 48-linkage specific polyubiquitin antibody. (B) Drosha is ubiquitinated in various cell types including HEK293T, HeLa and AGS. Left panel: HEK293T lysates were immunoprecipitated with control IgG or Rabbit Polyclonal Antibody to Drosha respectively. Right panel: Cell lysates were immunoprecipitated respectively with Rabbit Polyclonal Antibody to Drosha. The immunoprecipitates were resolved by SDS-PAGE and blotted with lysine 48-linkage specific polyubiquitin antibody. The same membrane was reblotted with Drosha Rabbit mAb. (C) Inhibition of the ubiquitin-proteasome pathway with MG132 (10 µM) increases endogenous Drosha protein level in AGS cells. GAPDH was used as a loading control. (D) Proteasomal inhibition increases exogenous GFP-Drosha expression level. HEK293T cells were transfected with GFP-Drosha. Twenty-four hours post-transfection, the cells were treated with 1 µM Clasto-Lactacystin-β-lactone (Omuralide) overnight. GFP-Drosha protein level was increased 3-fold following treatment.

Mentions: Previous studies have shown that acetylation inhibits ubiquitination of some proteins by competing for the same lysines [24], [25]. The linking of lysine 48 (K48) of ubiquitin to substrate proteins initiates proteasomal degradation and K48-linked polyubiquitin represents the active state of the ubiquitin-proteasome pathway [27], [28]. To determine the activity of ubiquitin-pathway, we first examined K48 linked polyubiquitin levels in a wide range of cells including HEK293T, Huh-7, AGS, MCF-7 and HeLa cells. We found that this pathway was constitutively active in all the cell lines tested (Figure 3A). We also found that Drosha was heavily ubiquitinated via K48-linked polyubiquitin (Figure 3B). Treatment of AGS cells with a specific proteasome inhibitor MG132 increased Drosha protein levels 3-fold (Figure 3C). Similar increases in Drosha protein were observed upon treatment of GFP-Drosha transfected HEK293T cells with the proteasome inhibitor Clasto-Lactacystin-β-lactone (Omuralide) (Figure 3D). All of these data indicate that ubiquitin-proteasome pathway is involved in Drosha degradation.


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)

A constitutively active ubiquitin-proteasome pathway degrades Drosha.Whole cell lysates of the indicated cell types were prepared with IP Lysis Buffer (Pierce); 20 µg protein aliquots were used for western blots. All experiments were repeated three times with similar results (p<0.05 by Student’s t-test). (A) Abundant polyubiquitination was observed in multiple cell types using lysine 48-linkage specific polyubiquitin antibody. (B) Drosha is ubiquitinated in various cell types including HEK293T, HeLa and AGS. Left panel: HEK293T lysates were immunoprecipitated with control IgG or Rabbit Polyclonal Antibody to Drosha respectively. Right panel: Cell lysates were immunoprecipitated respectively with Rabbit Polyclonal Antibody to Drosha. The immunoprecipitates were resolved by SDS-PAGE and blotted with lysine 48-linkage specific polyubiquitin antibody. The same membrane was reblotted with Drosha Rabbit mAb. (C) Inhibition of the ubiquitin-proteasome pathway with MG132 (10 µM) increases endogenous Drosha protein level in AGS cells. GAPDH was used as a loading control. (D) Proteasomal inhibition increases exogenous GFP-Drosha expression level. HEK293T cells were transfected with GFP-Drosha. Twenty-four hours post-transfection, the cells were treated with 1 µM Clasto-Lactacystin-β-lactone (Omuralide) overnight. GFP-Drosha protein level was increased 3-fold following treatment.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757024&req=5

pone-0072503-g003: A constitutively active ubiquitin-proteasome pathway degrades Drosha.Whole cell lysates of the indicated cell types were prepared with IP Lysis Buffer (Pierce); 20 µg protein aliquots were used for western blots. All experiments were repeated three times with similar results (p<0.05 by Student’s t-test). (A) Abundant polyubiquitination was observed in multiple cell types using lysine 48-linkage specific polyubiquitin antibody. (B) Drosha is ubiquitinated in various cell types including HEK293T, HeLa and AGS. Left panel: HEK293T lysates were immunoprecipitated with control IgG or Rabbit Polyclonal Antibody to Drosha respectively. Right panel: Cell lysates were immunoprecipitated respectively with Rabbit Polyclonal Antibody to Drosha. The immunoprecipitates were resolved by SDS-PAGE and blotted with lysine 48-linkage specific polyubiquitin antibody. The same membrane was reblotted with Drosha Rabbit mAb. (C) Inhibition of the ubiquitin-proteasome pathway with MG132 (10 µM) increases endogenous Drosha protein level in AGS cells. GAPDH was used as a loading control. (D) Proteasomal inhibition increases exogenous GFP-Drosha expression level. HEK293T cells were transfected with GFP-Drosha. Twenty-four hours post-transfection, the cells were treated with 1 µM Clasto-Lactacystin-β-lactone (Omuralide) overnight. GFP-Drosha protein level was increased 3-fold following treatment.
Mentions: Previous studies have shown that acetylation inhibits ubiquitination of some proteins by competing for the same lysines [24], [25]. The linking of lysine 48 (K48) of ubiquitin to substrate proteins initiates proteasomal degradation and K48-linked polyubiquitin represents the active state of the ubiquitin-proteasome pathway [27], [28]. To determine the activity of ubiquitin-pathway, we first examined K48 linked polyubiquitin levels in a wide range of cells including HEK293T, Huh-7, AGS, MCF-7 and HeLa cells. We found that this pathway was constitutively active in all the cell lines tested (Figure 3A). We also found that Drosha was heavily ubiquitinated via K48-linked polyubiquitin (Figure 3B). Treatment of AGS cells with a specific proteasome inhibitor MG132 increased Drosha protein levels 3-fold (Figure 3C). Similar increases in Drosha protein were observed upon treatment of GFP-Drosha transfected HEK293T cells with the proteasome inhibitor Clasto-Lactacystin-β-lactone (Omuralide) (Figure 3D). All of these data indicate that ubiquitin-proteasome pathway is involved in Drosha degradation.

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