title: Acetylation of drosha on the N-terminus inhibits its degradation by ubiquitination.
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.
authors.affiliation: Laboratory of Retrovirology, Division of Infectious Diseases, Department of Medicine, Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Tang
authors.forename: Sicheng
authors.initials: S
authors.lastname: Wen
authors.forename: Dong
authors.initials: D
authors.lastname: Zheng
authors.forename: Lynne
authors.initials: L
authors.lastname: Tucker
authors.forename: Lulu
authors.initials: L
authors.lastname: Cao
authors.forename: Dennis
authors.initials: D
authors.lastname: Pantazatos
authors.forename: Steven F
authors.initials: SF
authors.lastname: Moss
authors.forename: Bharat
authors.initials: B
authors.lastname: Ramratnam
mesh_terms: D000107:Acetylation; D002460:Cell Line; D057809:HEK293 Cells; D016481:Helicobacter Infections / Q000378:metabolism; D016480:Helicobacter pylori; D006801:Humans; D008239:Lysine / Q000378:metabolism; D035683:MicroRNAs / Q000096:biosynthesis; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D055550:Protein Stability; D059748:Proteolysis; D043244:Ribonuclease III / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D054875:Ubiquitination; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D014452:Ubiquitins; D043244:Ribonuclease III; D046988:Proteasome Endopeptidase Complex; D008239:Lysine