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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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N-terminus of Drosha is the main part for acetylation.(A) In vitro acetylation assays revealed that N’-terminal but not C’-terminal Drosha is the major acetylation domain. Immunoprecipitated N-terminal Drosha (GFP-Drosha1-390 ) or C-terminal Drosha (GFP-Drosha391-1374) was co-incubated with 10 U of p300 HAT Domain and 2 µM Acetyl CoA at 37°C for I hr. (B) Identification of an acetylated lysine site by mass spectrometry. (C) TSA treatment increases the acetylation and expression levels of GFP-DroshaK382R (D) miRNA sensor assays revealed that compared to vehicle control, treatment of cells with TSA increased miRNA function. (E) TSA treatment increases miR-143 level in HEK293T cells. (F) TSA treatment decreases expression of fibronectin type III domain containing 3B (FNDC3B), a target of miR-143. All experiments were performed in triplicate (p<0.05 by Student’s t-test).
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pone-0072503-g002: N-terminus of Drosha is the main part for acetylation.(A) In vitro acetylation assays revealed that N’-terminal but not C’-terminal Drosha is the major acetylation domain. Immunoprecipitated N-terminal Drosha (GFP-Drosha1-390 ) or C-terminal Drosha (GFP-Drosha391-1374) was co-incubated with 10 U of p300 HAT Domain and 2 µM Acetyl CoA at 37°C for I hr. (B) Identification of an acetylated lysine site by mass spectrometry. (C) TSA treatment increases the acetylation and expression levels of GFP-DroshaK382R (D) miRNA sensor assays revealed that compared to vehicle control, treatment of cells with TSA increased miRNA function. (E) TSA treatment increases miR-143 level in HEK293T cells. (F) TSA treatment decreases expression of fibronectin type III domain containing 3B (FNDC3B), a target of miR-143. All experiments were performed in triplicate (p<0.05 by Student’s t-test).

Mentions: To identify which part of Drosha is the target for acetylation, we created both a Drosha N-terminal construct (GFP-Drosha1-390) and a Drosha C-terminal construct (GFP-Drosha 391-1374). We transfected these two constructs into HEK293T cells separately and immunoprecipitated the N-terminal and C-terminal Drosha proteins. Using these immunoprecipitated Drosha proteins as substrates to incubate with purified p300 and acetyl CoA, we found that the N-terminal Drosha1-390, but not the C-terminal Drosha 391-1374 construct, harbored the likely lysine substrates for acetylation (Figure 2A). To identify which lysines were acetylated, we immunoprecipitated Drosha protein and analyzed protein modifications by mass spectrometry. We identified lysine 382 (K382) was acetylated (Figure 2B). However, when K382 was mutated, GFP-DroshaK382K could still be acetylated (Figure 2C), indicating that multiple lysines are likely to be involved in acetylation since there are 13 lysines on the N-terminal Drosha. We note that our ability to identify one acetylation site among the multiple lysines may be due to the possibility of ion suppression of the necessary fragmented ion during targeted MS/MS analysis. To determine if acetylation of Drosha affects microRNA functions, we used a miRNA-143 psiCHECK2 construct as described previously(21). We detected increased mature miRNA-143 as shown in the form of decreased Renila luciferase activity in HEK293T cells that were transfected with psiCHECK2 and then treated with 2 µM TSA for 6 hr (Figure 2D). To further examine the effects of Drosha acetylation on microRNA processing, miR-143 level was measured by real time PCR using a miR detection kit. Compared with vehicle control, TSA treatment increased miR-143 level significantly (Figure 2E). Previous study showed that miR-143 targets fibronectin to enhance hepatocarcinoma metastasis [26]. Our result showed that TSA decreased fibronectin type III domain containing 3B (FNDC3B) evidently (Figure 2F), suggesting that acetylation of Drosha is functional in microRNA biogenesis.


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)

N-terminus of Drosha is the main part for acetylation.(A) In vitro acetylation assays revealed that N’-terminal but not C’-terminal Drosha is the major acetylation domain. Immunoprecipitated N-terminal Drosha (GFP-Drosha1-390 ) or C-terminal Drosha (GFP-Drosha391-1374) was co-incubated with 10 U of p300 HAT Domain and 2 µM Acetyl CoA at 37°C for I hr. (B) Identification of an acetylated lysine site by mass spectrometry. (C) TSA treatment increases the acetylation and expression levels of GFP-DroshaK382R (D) miRNA sensor assays revealed that compared to vehicle control, treatment of cells with TSA increased miRNA function. (E) TSA treatment increases miR-143 level in HEK293T cells. (F) TSA treatment decreases expression of fibronectin type III domain containing 3B (FNDC3B), a target of miR-143. All experiments were performed in triplicate (p<0.05 by Student’s t-test).
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Related In: Results  -  Collection

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pone-0072503-g002: N-terminus of Drosha is the main part for acetylation.(A) In vitro acetylation assays revealed that N’-terminal but not C’-terminal Drosha is the major acetylation domain. Immunoprecipitated N-terminal Drosha (GFP-Drosha1-390 ) or C-terminal Drosha (GFP-Drosha391-1374) was co-incubated with 10 U of p300 HAT Domain and 2 µM Acetyl CoA at 37°C for I hr. (B) Identification of an acetylated lysine site by mass spectrometry. (C) TSA treatment increases the acetylation and expression levels of GFP-DroshaK382R (D) miRNA sensor assays revealed that compared to vehicle control, treatment of cells with TSA increased miRNA function. (E) TSA treatment increases miR-143 level in HEK293T cells. (F) TSA treatment decreases expression of fibronectin type III domain containing 3B (FNDC3B), a target of miR-143. All experiments were performed in triplicate (p<0.05 by Student’s t-test).
Mentions: To identify which part of Drosha is the target for acetylation, we created both a Drosha N-terminal construct (GFP-Drosha1-390) and a Drosha C-terminal construct (GFP-Drosha 391-1374). We transfected these two constructs into HEK293T cells separately and immunoprecipitated the N-terminal and C-terminal Drosha proteins. Using these immunoprecipitated Drosha proteins as substrates to incubate with purified p300 and acetyl CoA, we found that the N-terminal Drosha1-390, but not the C-terminal Drosha 391-1374 construct, harbored the likely lysine substrates for acetylation (Figure 2A). To identify which lysines were acetylated, we immunoprecipitated Drosha protein and analyzed protein modifications by mass spectrometry. We identified lysine 382 (K382) was acetylated (Figure 2B). However, when K382 was mutated, GFP-DroshaK382K could still be acetylated (Figure 2C), indicating that multiple lysines are likely to be involved in acetylation since there are 13 lysines on the N-terminal Drosha. We note that our ability to identify one acetylation site among the multiple lysines may be due to the possibility of ion suppression of the necessary fragmented ion during targeted MS/MS analysis. To determine if acetylation of Drosha affects microRNA functions, we used a miRNA-143 psiCHECK2 construct as described previously(21). We detected increased mature miRNA-143 as shown in the form of decreased Renila luciferase activity in HEK293T cells that were transfected with psiCHECK2 and then treated with 2 µM TSA for 6 hr (Figure 2D). To further examine the effects of Drosha acetylation on microRNA processing, miR-143 level was measured by real time PCR using a miR detection kit. Compared with vehicle control, TSA treatment increased miR-143 level significantly (Figure 2E). Previous study showed that miR-143 targets fibronectin to enhance hepatocarcinoma metastasis [26]. Our result showed that TSA decreased fibronectin type III domain containing 3B (FNDC3B) evidently (Figure 2F), suggesting that acetylation of Drosha is functional in microRNA biogenesis.

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