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. 2016 Jun 2;12(6):e1006095.
doi: 10.1371/journal.pgen.1006095. eCollection 2016 Jun.

Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein

Christian Much  1   2 , Tania Auchynnikava  3 , Dinko Pavlinic  4 , Andreas Buness  1 , Juri Rappsilber  3 , Vladimir Benes  4 , Robin Allshire  3 , Dónal O'Carroll  1   2
Affiliations

Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein

Christian Much et al. PLoS Genet. .

Abstract

Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Dicer localizes exclusively to the cytoplasm of embryonic fibroblasts and adult tissues.
(A) Schematic representation of the domain structure of N-terminal Flag-HA2 tagged murine Dicer. (B) Western blot using anti-HA antibody on serial dilution of whole cell extract from DcrFH/FH PMEFs. (C, E and G) Western blot analysis after subcellular fractionation using anti-HA antibody on cytoplasmic (Cy), nuclear (Nu) and whole cell (WC) extracts from wild type and DcrFH/FH PMEFs (C), adult testis (E) and thymus (G). Tubulin served as a cytoplasmic marker and histone H3 as a nuclear marker. (D, F and H) Confocal immunofluorescence micrographs showing wild type and DcrFH/FH PMEFs (D) as well as sections of adult testis (F) and thymus (H) stained with anti-HA antibody. Nuclei are stained with Hoechst. Scale bars represent 5 μm. (I) Immunoprecipitation using anti-HA beads and mass spectrometry analysis of the Dicer interactome in PMEFs, testis and thymus (each biological duplicates) and all of them pooled (six biological replicates).
Fig 2
Fig 2. Dicer is not a nucleocytoplasmic shuttling protein.
(A) Confocal micrographs showing immunofluorescence anti-HA staining of wild type and DcrFH/FH PMEFs without and with LMB-induced inhibition of nuclear export. Cells were treated with 20 nM LMB or solvent for 6 h. Cyclin B1 served as a positive control for the LMB treatment. Nuclei are stained with Hoechst. Scale bars represent 10 μm. (B) Western blot analysis after LMB treatment and subcellular fractionation. Anti-HA antibody was used on cytoplasmic (Cy) and nuclear (Nu) extracts, where tubulin served as a cytoplasmic marker and histone H3 as a nuclear marker.
Fig 3
Fig 3. FGF signaling does not induce translocation of Dicer to the nucleus.
(A) Confocal micrographs showing immunofluorescence anti-HA staining of wild type and DcrFH/FH serum starved PMEFs after stimulation with FGF2. Nuclei are stained with Hoechst. Scale bars represent 10 μm. (B) Western blot analysis after FGF2 stimulation and subcellular fractionation. Anti-HA antibody was used on cytoplasmic (Cy) and nuclear (Nu) extracts of treated and untreated cells. Tubulin is a cytoplasmic marker and histone H3 a nuclear marker. (C and D) Anti-HA immunofluorescence of wild type and DcrFH/FH uterus sections showing epithelial cells lining the lumen (C) and uterine glands (D).
Fig 4
Fig 4. Dicer does not translocate to the nucleus after DNA damage.
(A) Confocal imaging of anti-HA stained wild type and DcrFH/FH PMEFs after irradiation. Cells were irradiated with 20 Gy and then allowed to recover for 30 min. Activation of the DNA damage response was verified with anti-γH2AX staining. Nuclei are stained with Hoechst. Scale bars represent 10 μm. (B) Western blot analysis after irradiation and subcellular fractionation. Anti-HA antibody was used on cytoplasmic (Cy) and nuclear (Nu) extracts of irradiated and non-irradiated cells. Tubulin served as a cytoplasmic marker, histone H3 as a nuclear marker and γH2AX as a marker of DNA damage. (C) Confocal microscopy on anti-HA stained wild type and DcrFH/FH PMEFs after irradiation and LMB treatment.
Fig 5
Fig 5. Dicer is solely cytoplasmic during embryogenesis.
(A) Anti-HA immunofluorescence of E13.5 DcrFH/FH embryo section. Scale bar is 1 mm. (B-I) Confocal microscopy of immunofluorescence staining of wild type and DcrFH/FH E13.5 tissue sections with anti-HA antibody. Sections of lung (B) and liver (C) are shown as representatives of endoderm, vertebrae (D) represent mesoderm, forebrain (E), root ganglion (F), lens (G) and epidermis (H) represent ectoderm, placenta (I) is depicted as an example for an extraembryonic tissue. Nuclei are stained with Hoechst. Scale bar is 10 μm.
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References

    1. Cerutti H, Casas-Mollano JA (2006) On the origin and functions of RNA-mediated silencing: from protists to man. Curr Genet 50: 81–99. - PMC - PubMed
    1. O'Carroll D, Schaefer A (2013) General principals of miRNA biogenesis and regulation in the brain. Neuropsychopharmacology 38: 39–54. 10.1038/npp.2012.87 - DOI - PMC - PubMed
    1. Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, et al. (2002) Prediction of plant microRNA targets. Cell 110: 513–520. - PubMed
    1. Tang G, Reinhart BJ, Bartel DP, Zamore PD (2003) A biochemical framework for RNA silencing in plants. Genes Dev 17: 49–63. - PMC - PubMed
    1. Guo H, Ingolia NT, Weissman JS, Bartel DP (2010) Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466: 835–840. 10.1038/nature09267 - DOI - PMC - PubMed

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