Phosphorylation of the RNase III enzyme Drosha at Serine300 or Serine302 is required for its nuclear localization

Laboratory of Retrovirology, Division of Infectious Diseases, Department of Medicine, Warren Alpert Medical School of Brown University, Providence, RI 02903, USA.
Nucleic Acids Research (Impact Factor: 9.11). 10/2010; 38(19):6610-9. DOI: 10.1093/nar/gkq547
Source: PubMed


The RNaseIII enzyme Drosha plays a pivotal role in microRNA (miRNA) biogenesis by cleaving primary miRNA transcripts to generate
precursor miRNA in the nucleus. The RNA binding and enzymatic domains of Drosha have been characterized and are on its C-terminus.
Its N-terminus harbors a nuclear localization signal. Using a series of truncated Drosha constructs, we narrowed down the
segment responsible for nuclear translocation to a domain between aa 270 and aa 390. We further identified two phosphorylation
sites at Serine300 (S300) and Serine302 (S302) by mass spectrometric analysis. Double mutations of S→A at S300 and S302 completely
disrupted nuclear localization. Single mutation of S→A at S300 or S302, however, had no effect on nuclear localization indicating
that phosphorylation at either site is sufficient to locate Drosha to the nucleus. Furthermore, mimicking phosphorylation
status by mutating S→E at S300 and/or S→D at S302 restored nuclear localization. Our findings add a further layer of complexity
to the molecular anatomy of Drosha as it relates to miRNA biogenesis.

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    • "Our previous studies showed that GSK3β phosphorylates Drosha, the key RNase III enzyme that initiates microRNA (miR) biogenesis (9,10). MiRs are transcribed into primary miRs (pri-miRs) from miR genes by polymerase II or III. "
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    ABSTRACT: Glycogen synthase kinase 3 beta (GSK3β) is a critical protein kinase that phosphorylates numerous proteins in cells and thereby impacts multiple pathways including the β-Catenin/TCF/LEF-1 pathway. MicroRNAs (miRs) are a class of noncoding small RNAs of ∼22 nucleotides in length. Both GSK3β and miR play myriad roles in cell functions including stem cell development, apoptosis, embryogenesis and tumorigenesis. Here we show that GSK3β inhibits the expression of miR-96, miR-182 and miR-183 through the β-Catenin/TCF/LEF-1 pathway. Knockout of GSK3β in mouse embryonic fibroblast cells increases expression of miR-96, miR-182 and miR-183, coinciding with increases in the protein level and nuclear translocation of β-Catenin. In addition, overexpression of β-Catenin enhances the expression of miR-96, miR-182 and miR-183 in human gastric cancer AGS cells. GSK3β protein levels are decreased in human gastric cancer tissue compared with surrounding normal gastric tissue, coinciding with increases of β-Catenin protein, miR-96, miR-182, miR-183 and primary miR-183-96-182 cluster (pri-miR-183). Furthermore, suppression of miR-183-96-182 cluster with miRCURY LNA miR inhibitors decreases the proliferation and migration of AGS cells. Knockdown of GSK3β with siRNA increases the proliferation of AGS cells. Mechanistically, we show that β-Catenin/TCF/LEF-1 binds to the promoter of miR-183-96-182 cluster gene and thereby activates the transcription of the cluster. In summary, our findings identify a novel role for GSK3β in the regulation of miR-183-96-182 biogenesis through β-Catenin/TCF/LEF-1 pathway in gastric cancer cells.
    Full-text · Article · Dec 2013 · Nucleic Acids Research
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    • "Modulation of protein stability by phosphorylation is becoming a common theme in biology, and examples of crosstalk between phosphorylation and ubiquitin-mediated degradation of proteins are increasingly being reported (Hunter, 2007). Within the miRNA biogenesis pathway itself, changes in the PTMs of miRNA processing enzymes and their dsRNA-binding partners, effected by cell-signaling pathways, have been reported for TRBP2 and Drosha phosphorylation, and for DGCR8 and Drosha acetylation (Paroo et al., 2009; Tang et al., 2010, 2011, 2013; Wada et al., 2012). Exactly how phosphorylation confers increased stability to DGCR8 or TRBP2 is not yet known. "
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    ABSTRACT: During miRNA biogenesis, the microprocessor complex (MC), which is composed minimally of Drosha, an RNase III enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary miRNA (pri-miRNA) in order to release the pre-miRNA stem-loop structure. Using phosphoproteomics, we mapped 23 phosphorylation sites on full-length human DGCR8 expressed in insect or mammalian cells. DGCR8 can be phosphorylated by mitogenic ERK/MAPK, indicating that DGCR8 phosphorylation may respond to and integrate extracellular cues. The expression of phosphomimetic DGCR8 or inhibition of phosphatases increased the cellular levels of DGCR8 and Drosha proteins. Increased levels of phosphomimetic DGCR8 were not due to higher mRNA levels, altered DGCR8 localization, or DGCR8's ability to self-associate, but rather to an increase in protein stability. MCs incorporating phosphomutant or phosphomimetic DGCR8 were not altered in specific processing activity. However, HeLa cells expressing phosphomimetic DGCR8 exhibited a progrowth miRNA expression profile and increased proliferation and scratch closure rates relative to cells expressing phosphomutant DGCR8.
    Full-text · Article · Nov 2013 · Cell Reports
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    • "AGS cells were transfected with GenJet Plus DNA Transfection Reagent (SignaGen Laboratories) when cell confluency was ∼70%. GFP-Drosha construct has been described previously [21] and was used as a backbone for all truncated and mutated constructs. All mutants were constructed by means of site-directed mutagenesis with pfu DNA polymerase (Stratagene) as previously described [21], [22]. "
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    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.
    Full-text · Article · Aug 2013 · PLoS ONE
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