The authors have declared that no competing interests exist.
Dicer is a key component of RNA interference (RNAi) and well-known for its role in biogenesis of micro (mi)RNA in the cytoplasm. Increasing evidence suggests that mammalian Dicer is also present and active in the nucleus. We have previously shown that phosphorylated human Dicer associates with chromatin in response to DNA damage and processes double-stranded (ds)RNA in the nucleus. However, a recent study by Much et al. investigated endogenously tagged HA-Dicer both in primary mouse embryonic fibroblasts (PMEFs) as well as adult homozygous viable and fertile HA-Dicer mice under physiological conditions and concluded that murine Dicer is exclusively cytoplasmic. The authors challenged several findings, reporting functions of Dicer in mammalian nuclei. We have re-investigated this issue by applying subcellular fractionation, super-resolution microscopy followed by 3D reconstitution, and phospho-Dicer-specific antibodies using the same HA-Dicer PMEF cell line. Our data show that a small fraction of the murine HA-Dicer pool, approximately 5%, localises in the nucleus and is phosphorylated upon DNA damage. We propose that Dicer localisation is dynamic and not exclusively cytoplasmic, particularly in cells exposed to DNA damage.
Cytoplasmic Dicer is a key component of the canonical micro (mi)RNA biogenesis pathway. However, a growing body of evidence points toward localisation and activity of mammalian Dicer in the nucleus. A recent study by Much et al., employed an endogenously HA-tagged Dicer knock-in mouse cell line to show that Dicer is exclusively cytoplasmic. This paper challenges several studies reporting various RNA metabolic functions of Dicer in human nuclei. Given the controversy about Dicer’s subcellular localisation, it is essential to address this issue. Employing the same cells as used by Much and colleagues, we combined super-resolution microscopy followed by 3D reconstitution and biochemical assays to show that endogenously tagged HA-Dicer prominently localises in the nucleus under physiological conditions. We demonstrate that DNA damage triggers accumulation of phosphorylated HA-Dicer in the nucleus, confirming previous observations in human cells. Our data indicate evolutionary conservation of nuclear Dicer localisation and function in mammals in response to DNA damage.
The endoribonuclease Dicer recognises and processes double-stranded (ds)RNA substrates of various origins into small non-coding (nc)RNA [
However, mechanistic insight in mammalian nuclear Dicer localisation remains largely inconclusive. Analysis of ectopically expressed human Dicer mutants suggest that the dsRNA binding domain (dsRBD) may harbour a cryptic nuclear localisation signal, which is potentially occluded by the helicase domain in the full-length Dicer protein [
Moreover, recent work by Much et al. challenged the existence of mammalian Dicer in the nucleus
Here, we provide evidence for existence of HA-Dicer in murine nuclei under physiological conditions and involvement of nuclear phosphorylated HA-Dicer in the DDR. Using subcellular fractionation, super-resolution microscopy followed by 3D reconstitution and phospho-Dicer-specific antibodies, we demonstrate that a small fraction of HA-Dicer localises to nuclei of unperturbed cells. Following DNA damage, phosphorylated HA-Dicer accumulates in the nucleus in a phosphatidylinositol-3-kinase (PI3K)-dependent manner. We propose that a subset of the mammalian Dicer pool relocalises to the nucleus rather than being exclusively restricted to the cytoplasm.
Comprehensive assessment of subcellular localisation of endogenously tagged HA-Dicer in PMEF::HA-Dicer cells (
(A) Schematic of endogenously tagged Dicer (HA-Dicer) in primary mouse embryonic fibroblast (PMEF::HA-Dicer) cells (not in scale). HA, epitope tag; DEXD/H, HELICc, helicase domain; DUF283, domain of unknown function; PAZ, Piwi/Argonaute/Zwille; RIIIa/b, RNaseIII a/b; dsRBD, double-stranded RNA binding domain. HA-Dicer is expressed from a single endogenous murine
Previous investigations of HA-Dicer in PMEF::HA-Dicer cells excluded any nuclear localisation or activity of HA-Dicer [
(A) Immunoblot displaying reactivity of phospho-Dicer antibodies p-DCR-1 (p-DCR-1, mixture of two individual antibodies recognising Ser1712 or Ser1836 individually, see also section “
Confocal imaging of interspecies heterokaryon fusions between human HEK293 cells and murine PMEF::HA-Dicer or wild type PMEF cells, respectively. Heterokaryons were stained for total HA-Dicer (3F10), γH2A.X in absence or presence of Etoposide. Cytoskeleton was stained using Alexa Fluor 647-conjugated Phalloidin. Nuclei:
Next, we tested for specificity and sensitivity of HA antibodies in immunofluorescence microscopy. We co-cultured wild type PMEFs and PMEF::HA-Dicer cells in absence or presence of nuclear export inhibitor LMB or DNA damage-inducing Topoisomerase II inhibitor Etoposide prior to HA staining with either C29F4, HA.11 or 3F10 antibody (
To reassess the subcellular localisation of HA-Dicer in unperturbed PMEF::HA-Dicer cells, we combined the 3F10 HA antibody with highly sensitive super-resolution microscopy. We detected clear nuclear 3F10 staining, in addition to prominent reactivity in the cytoplasm (
To estimate the amount of nuclear HA-Dicer in the nucleus of unperturbed cells quantitatively, we measured nuclear HA-Dicer band intensities relative to cytoplasmic levels and calculated the amount of nuclear Dicer in % normalised to a cytoplasmic-to-nucleoplasmic input ratio of 1:3, reflecting a 3-fold concentrated nuclear fraction. Values for nuclear HA-Dicer varied between 3.7% and 9.7%, depending on the HA antibody. We conclude that a subset of approximately 5% of the total HA-Dicer pool localises to the nucleus of PMEF::HA-Dicer cells under physiological conditions.
We have recently discovered that a subset of human Dicer is phosphorylated in response to DNA damage to process damage-induced dsRNA in the nucleus [
Next, we preformed subcellular fractionation of PMEF::HA-Dicer cells cultured in presence or absence of Etoposide (
To visualise phosphorylated HA-Dicer, we performed confocal imaging of PMEF::HA-Dicer cells stained with p-DCR-1 antibodies in absence or presence of Etoposide (
Three members of the phosphatidylinositol-3-kinase (PI3K) family—ATM, ATR and DNA-dependent protein kinase (DNA-PK)—govern the response to DNA damage by phosphorylating hundreds of substrates [
To further assess the subcellular localisation of phosphorylated HA-Dicer in PMEF::HA-Dicer cells in response to DNA damage, we preformed γ-irradiation. We have recently described damage-induced nuclear localisation of phosphorylated human Dicer 2–3 hours after γ-irradiation with a total dose of 10 Gray (Gy) [
To further validate specificity of 3F10 HA and p-DCR-1 antibodies in confocal imaging, we repeated γ-irradiation in both wild type PMEFs and Dicer-/- MEFs at optimised conditions. As expected, γ-irradiation neither induced 3F10 reactivity in wild type PMEFs (
For proof of principle, we performed an interspecies heterokaryon experiment (
Taken together, our data indicate that a subset of HA-Dicer localises to nuclei of unperturbed cells, with increased levels of phosphorylated HA-Dicer being detectable in damaged nuclei. We show that phosphorylation of HA-Dicer in response to DNA damage triggers accumulation of HA-Dicer in the nucleus. Phosphorylated nuclear HA-Dicer arguably reflects a minor fraction of the HA-Dicer pool, which we estimate to be 5%. Importantly, no adverse impact on viability and fertility of mice homozygous for the
Recent evidence from
We conclude that Dicer proteins are found in the nuclei of the vast majority of studied eukaryotes, including mammals. The cytoplasm remains the main compartment for Dicer localisation. However, during development or stress, a subset of the cytoplasmic Dicer pool may be altered either genetically, by proteolysis, heat shock, or by PTMs to adjust for Dicer subcellular localisation or activity, suggesting structural and functional distinct nuclear Dicer subpopulations. Our findings point toward additional layers of complexity in the regulation of RNAi components and underscore the relevance of studying mechanisms of non-canonical RNAi in mammals.
Wild type or HA epitope-tagged primary mouse embryonic fibroblasts (PMEF wt and PMEF::HA-Dicer, female, a kind gift from the O’Carroll Lab), or Dicer knockout mouse embryonic fibroblasts (MEF Dicer-/-, clone [1A11], [
Subcellular fractionation was performed as described [
The amount of nuclear Dicer relative to cytoplasmic levels and normalised to the 1:3 input ratio was calculated in % using the following equation: [(HA Ab signal in nuclear fraction / HA Ab signal in cytoplasmic fraction) / (7.5% of lysed nuclei / 2.5% of lysed cytoplasm)] x 100%. Intensities of bands were quantified using ImageJ and values for nuclear Dicer were plotted as relative signals normalised to signals from cytoplasmic fractions or non-damaged nuclear fractions, respectively. For example, calculation of amount of nuclear Dicer using 3F10 Ab as shown in
Whole cell extracts from approximately 5x 105 cells grown on 6-well multi-well dishes (Corning) were lysed directly in 100 μl 4 x SDS-PAGE sample buffer, 10 μl of lysate was loaded, separated and stained with Ponceau S (Sigma) prior to antibody hybridisation. For semi-quantitative analysis of detection thresholds of HA antibodies, 10 μl lysate of PMEF::HA-Dicer cells, which were grown on 6-well multi-well dishes and lysed in 100 μl 4 x SDS-PAGE sample buffer, was diluted 5 times in a 2-fold serial dilution series. HA signal intensities of 10 μl of either non-diluted sample, i.e. input, or diluted samples, or Ponceau S stainings thereof, were quantified using ImageJ and plotted as relative normalised signals. Signals were plotted, loss of reactivity of HA antibodies was visualised as gap and quantified as delta (Δ = Rel. norm. Ponc. S signal—Rel. norm. HA Ab signal). For each HA antibody, a Δ was calculated at dilutions steps 1:2 or 1:4 to quantify signals in the near-linear range of sensitivity.
For immunoprecipitation, approximately 3x 106 wild type PMEF or PMEF::HA-Dicer cells grown on 10 cm dishes (Corning) were trypsinised, washed in cold 1x PBS and centrifuged (1200rpm, 5 min). Pellets were lysed in 5 volumes WCE lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% NP-40, 2 mM MgCl2, 50 mM NaF, 1 x protease/phosphatase inhibitor cocktails, Roche) for 20 minutes on ice, sonicated and Benzonase digested as described above. WCE lysates were precleared with protein G agarose beads (Merck Millipore) for 30 min. Samples were incubated with 5 ug primary HA antibodies for 4 hours and pulled down using protein G agarose beads for 45 min. IP samples were washed three times for 10 min with 800 μl WCE lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% NP-40, 2 mM MgCl2, 50 mM NaF, 1 x protease/phosphatase inhibitor cocktails, Roche), and eluted with SDS-PAGE sample buffer.
The following primary antibodies were used: anti-α-Tubulin (Abcam, [YL1/2], ab6160); anti-Rad21 (Merck Millipore, 05–908); anti-RNAPII-CTD S2P (Abcam, ab5095); anti-histone H3 (Abcam, ab1791); anti-HA (Roche, [3F10], 11867423001); anti-HA (BioLegend, [16B12], 901501, previously Covance MMS-101P); anti-HA (CST, [C29F4], 3724); anti-Lamin C (Novus, [EM11], NBP1-50051); anti-KDEL motif (Abcam, [10C3], ab12223); anti-γH2A.X (Ser139, Merck Millipore, 05–636); anti-Drosha (Abcam, ab12286); anti-Cyclin E (Santa Cruz, [M-20], sc-481); anti-Cyclin B1 (Abcam, ab2949); anti-c-Myc (Clontech, 631206); anti-53BP1 (Santa Cruz, [H-300], sc-22760); anti-pATM/ATR substrates mix (CST, [SxQ, D23H2/D69H5], 9670); anti-pChk1 (Ser345, CST, 133D3); anti-pChk1 (Ser317, CST, D12H3); anti-pERK1/2 (Thr202/Tyr204, CST, 9101); anti-ERK1/2 (CST, 9102); anti-p-p38 (Thr180/Tyr182, CST, 9211); anti-p21 (Santa Cruz, [C-19], sc-397); anti-p16 (Santa Cruz, [F-12], sc-1661); and anti-p-DCR-1 (Ser1712/Ser1836, a kind gift from the Arur Lab) [
The p-DCR-1 signals represent a mixture of two individual antibodies, raised against carboxy-terminal murine Dicer epitopes phospho-Ser1712 and phospho-Ser1836 individually in separate rabbits. Murine epitopes Ser1712/Ser1836 are equivalent to human epitopes Ser1728/Ser1852 and
Approximately 3x 105 wild type PMEFs, Dicer-/- MEFs clone [1A11], and PMEF::HA-Dicer cells grown on 6-well multi-well dishes (Corning) were washed in 1x PBS, fixed on coverslips with 3% Paraformaldehyde in PBS for 10 min, washed and incubated with 50 mM Ammonium chloride in PBS for 10 min, washed in 1x PBS, permeabilised with PBS/0.1% Tween for 5 min and blocked with PBS/10% FBS for 2 hours at 4°C. Primary antibodies were incubated overnight at 4°C in PBS/0.15% FBS. Alexa Flour 488-, 555-, or 647-conjugated secondary antibodies (Invitrogen) were incubated in PBS/0.15% FBS at room temperature for 1.5 hours in a humidified chamber. Cells were washed 3 times for 5 minutes with PBS/0.1% Triton-X 100 between antibody incubations. Nuclei were counterstained and mounted with 6-diamidino-2-phenylindole (DAPI)-containing Mowiol (Merck Millipore).
For confocal imaging, slides were processed on an Olympus microscope, using 60x lens. Samples with 1.5-mm coverslips were imaged using a an FV1000 confocal system on an Olympus IX-81 microscope with photomultiplier tube detectors and Olympus PlanApo N, 60×/1.35NA lens at RT. DAPI-containing Mowiol (EMD Millipore) was used as the imaging medium. DAPI; Alexa Fluor 488, 539, and 635 (Thermo Fisher Scientific) channels were used for acquisition with Olympus Fluoview software. Experimental settings including values of the laser power for each channel, HV, gain and offset parameters were determined at the beginning of each individual imaging session (by assessing background reactivity and saturation levels of each channel) and kept constant over the entire imaging session. ImageJ software (NIH) was used for further processing of the images. All quantifications represent a number of cells that have shown phenotype or % of positive cells, see figure legends for details. n = number of cells. Signals were quantified using RGB profiler (ImageJ, NIH). For super-resolution microscopy, cells were imaged at room temperature using an inverted Zeiss 880 microscope fitted with an airy-scan detector. The system was equipped with Plan- Apochromat 63x/1.4 NA oil lens. 488 nm argon and 405nm, and 633 nm diode lasers were used to excite GFP, DAPI and Alexa Fluor 633, respectively. Sequential excitation of each wavelength was switched per line to ensure blue, green and red channels were aligned. Sections of 20 slices with 0.5 μm thick intervals were collected with a zoom value of 600 pixels/μm. Images were processed using Airyscan processing (Zeiss 880 Airyscan: Airyscan is a special detector added to the coupling port of LSM 880. It is more light efficient than a standard confocal point detector. The extra on photons can be used to increase the sensitivity of the image, to scan faster or to improve the resolution) in 3D with a strength value of
Approximately 3x 105 murine wild type PMEF or PMEF::HA-Dicer cells were grown to 70–80% confluency on 6-well multi-well dishes (Corning). Approximately 2x 105 wild type human HEK293 cells were seeded on top of the PMEF layer prior to membrane fusion. Mixed cell populations were grown in presence of Cycloheximide (50 μg/ml) for 4 hours prior to fusion. For heterokaryon formation, cells were washed with warm 1x PBS, incubated with 100 μl warm PEG-3000 solution (50% w/v in PBS) for 2 minutes and washed with 1x PBS five times. Heterokaryons were cultured for additional 2 hours in Cycloheximide- containing medium in presence or absence of Etoposide (25 μM) prior to fixation. Alexa Fluor 647-conjugated Phalloidin (Life Technology) was used to stain the cytoskeleton. Preparation of immunofluorescence slides was performed as described in section imaging analysis.
Immunoblots probing for cyclin E, cyclin B1, c-Myc, as well as total and phosphorylated ERK1/2 kinases, phosphorylated p38 kinase, or cell cycle inhibitors p21 and p16 in PMEF::HA-Dicer whole cells extracts following starvation (0.1% fetal bovine serum, FBS) or serum stimulation (0.1% FBS +20% FBS). Control, 10% FBS; Rad21, cohesin subunit, loading control; #, unspecific signal.
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(A and B) Immunoblots displaying reactivity of HA antibodies C29F4, HA.11, 3F10 following incubation with (A) whole cell extracts of wild type PMEF, PMEF::HA-Dicer or Dicer-/- knockout MEF cells (clone 1A11) and with (B) serial dilution of PMEF::HA-Dicer whole cell extract. HA signals were quantified as arbitrary units relative to Ponceau S staining and normalised to input (IN, undiluted whole cell extract, 10% of lysate is loaded). Loss of reactivity of HA antibodies is defined as deltaHA (ΔHA = Rel. norm. Ponc. S signal—Rel. norm. HA Ab signal). Values at non-diluted input samples were set to 1. Ponceau S, loading control; #, aberrant signals; Ab, antibody. See also section “
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(A) Confocal imaging of wild type PMEF and PMEF::HA-Dicer co-cultures using HA antibodies C29F4, HA.11 and 3F10 in absence or presence of Leptomycin B (LMB) or Etoposide. Representative merged images are shown.
(B) Immunoblots displaying reactivity of HA antibodies C29F4, HA.11 and 3F10 incubated with whole cell extracts of PMEF::HA-Dicer cells following treatment with Leptomycin B (LMB) or Etoposide. #, aberrant signals; Ab, antibody.
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(A) Immunoblots detecting substrates of Ataxia-telangiectasia mutated (ATM) and ATM-related (ATR) kinase activity (pATM/ATR substrates mix antibody), p21 and phosphorylated histone variant H2A.X (γH2A.X, Ser139) levels in PMEF::HA-Dicer whole cell extracts following γ-irradiation (10 Gy, 2 hours recovery time) or incubation with Etoposide or Leptomycin B (LMB). (B) Immunoblots displaying reactivity of HA antibody 3F10 and γH2A.X levels after incubation with whole cell extracts (IN, input, 10% of lysate is loaded) of wild type PMEFs or PMEF::HA-Dicer cells or after immunoprecipitation (IP) using 3F10 antibody in absence or presence of Etoposide. IgG, immunoglobulin heavy chain, loading control. (C) Immunoblot detecting phosphorylated HA-Dicer using p-DCR-1 antibodies following IP with 3F10 antibody as described in (B); #, aberrant signal; M, molecular-weight size marker. (D) Immunoblots displaying reactivity of p-DCR-1 antibodies following incubation with whole cell extracts of wild type PMEFs, PMEF::HA-Dicer cells or Dicer-/- knockout MEFs after treatment with Etoposide. H3, histone 3, loading control; #, unspecific signal. (E) Immunoblots detecting total HA-Dicer (HA.11, 3F10) in subcellular fractions of PMEF::HA- Dicer cells in absence or presence of Etoposide. H3, histone H3; CP, cytoplasm; NP, nucleoplasm; CP and NP fractions are loaded in a 1:1 ratio. See also section “
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(A) Confocal imaging of phosphorylated HA-Dicer (p-DCR-1) and total HA-Dicer (3F10) in PMEF::HA-Dicer cells in presence or absence of Etoposide or after preincubation with Phosphatidylinositol-3-kinase (PI3K) inhibitors. Quantitation indicates cells with shown phenotype in % and number of cell analysed (n) (top panel). RGB profiles of p-DCR-1 (green) and HA-Dicer (red and blue) signals in representative cells. (B) Immunoblots detecting total HA-Dicer (3F10, HA.11, C29F4) as well as levels of phosphorylated checkpoint kinase 1 (pChk1, Ser317/Ser345) and γH2A.X in PMEF::HA-Dicer cells in presence or absence of Etoposide or after preincubation with Phosphatidylinositol-3-kinase (PI3K) inhibitors. Ponceau S, loading control.
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Time course confocal imaging of PMEF::HA-Dicer cells stained for phosphorylated Dicer (p-DCR-1), γH2A.X and total HA-Dicer (3F10) following γ-irradiation with a total dose of 10 Gy and recovery for various hours. Quantitation indicates cells with shown phenotype in % and number of cell analysed (n).
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(A) Confocal imaging of wild type PMEF cells stained for phosphorylated Dicer (p-DCR-1), γH2A.X and total HA- Dicer (3F10) following γ-irradiation with a total dose of 10 Gy and 2 hours recovery time.
(B) as in (A), but using Dicer-/- MEFs (clone 1A11). (C) Confocal imaging of PMEF::HA- Dicer cells incubated with Alexa-Flour conjugated secondary antibodies. (D) Confocal imaging of wild type PMEF (
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(A) Immunoblot detecting phosphorylated HA-Dicer (p-DCR-1) in PMEF::HA-Dicer whole cell extracts in presence or absence of γ-irradiation (left) and immunoblots displaying reactivity of p-DCR-1 antibodies following incubation with whole cell extracts of wild type PMEFs, PMEF::HA-Dicer cells or Dicer-/- knockout MEFs upon γ-irradiation (right); H3, histone 3, loading control; #, unspecific signal. (B and C) RGB profiles (B) and 3D reconstitution of super-resolution microscopy imaging (C) for phosphorylated HA-Dicer (p-DCR-1, green) or total HA-Dicer (3F10, red) in non-irradiated (Control) cells or cells irradiated with 10 Gy followed by 0.5–5 hour recovery time. Representative images are shown.
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We thank Alain Wainman and Micron facility for technical assistance on imaging analysis. We are grateful to Swathi Arur’s lab for generous supply with p-DCR-1 antibodies and Donal O’Carroll for providing us with primary mouse embryonic fibroblast cells as well as Vigo Heissmeyer for providing Dicer knockout MEF cells. We further acknowledge members of Ivan Ahel’s, Dragana Ahel’s, Fumiko Esashi’s, and Matthew Freeman’s groups for their help, reagents and valuable comments, as well as members of the M.G. lab for a creative atmosphere and technical support.