1. Chromosomes and Gene Expression
  2. Stem Cells and Regenerative Medicine
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MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response

  1. Jeroen Witteveldt
  2. Lisanne I Knol
  3. Sara Macias  Is a corresponding author
  1. University of Edinburgh, United Kingdom
Research Article
Cite this article as: eLife 2019;8:e44171 doi: 10.7554/eLife.44171
8 figures and 2 additional files

Figures

Figure 1 with 1 supplement
ESCs lack IFN response and are more susceptible to viral infection.

(a) Quantification of Ifnb1 expression in ESCs and the somatic mouse cell lines NIH3T3 and BV-2 after transfection with the dsRNA analogue poly(I:C). Data show the average (n = 3)±s.e.m, (*) p-value<0.05 by t-test. (b) Quantification of Ifnb1 expression after activation of the cGAS response by Y-DNA (G3-YSD) in the same cells lines as (a). Data show the average (n = 3, except for ESC2, n = 2)±s.e.m, (*) p-value<0.05 by t-test. (c) Susceptibility (TCID50/ml) of same cell lines as used in (a) to TMEV infection. (d) Quantification of Ifnb1 expression in pluripotent and differentiated ESCs after activation with poly(I:C). Data show the average (n = 3) fold change over mock treated cells,±s.d. (*) p-value<0.05 by t-test.

https://doi.org/10.7554/eLife.44171.003
Figure 1—figure supplement 1
Retinoic acid differentiation of ESCs.

(a) Quantification of pluripotency markers, Nanog and Pou5f1 (Oct-4) expression by qRT-PCR upon differentiation with retinoic acid (RA), and normalized to Actb expression, during pluripotency (ESC), after 2 days in hanging droplets, 2 days (2d RA) and 10 days in retinoic acid (10d RA) (b) Quantification of differentiation markers expression, Neurog2, Gata6 and Gata4 upon differentation with retinoic acid, as in (a). Both panels include data of one representative experiment.

https://doi.org/10.7554/eLife.44171.004
Figure 2 with 2 supplements
MiRNAs regulate IFN response.

(a) (left) Susceptibility (TCID50/ml) of miRNA deficient cells (Dgcr8-/-, Dicer-/-) and wild-type parental cells (Dgcr8+/+(ESC1), Dicer+/+(ESC2)) to TMEV infection, higher values represent higher susceptibility (n = 4, p-value<0.05, t-test). (right) Quantification of Influenza A replication after infection of the same cell lines, data show the average (n = 3)±s.d. (*) p-value<0.05 by t-test. (b) Quantification of Ifnb1 expression of ESCs lacking Dgcr8 or Dicer to stimulation with poly(I:C) and G3-YSD. Data show average (n = 3)±s.d., normalized to mock, (*) p-value<0.05 by t-test. (c) Quantification of TMEV vRNA upon JAK1/2 inhibition by Ruxolitinib treatment. Data show the average (n = 3)±s.d. (d) qRT-PCR analyses of ISGs expression after stimulation of wild-type and miRNA-deficient ESCs with IFN-β. Data show average (n = 3)±s.d., normalized to mock treated cells (e, f) Quantification of TMEV replication after infection in Dgcr8 (e) and Dicer (f) parental (+/+), deficient (-/-) and rescued (resc) cell lines. Data are normalized to miRNA-deficient cell lines susceptibility. Data show the average (n = 3)±s.d (*) p-value<0.05 by t-test. Northern blots for three stem-cell specific miRNAs, as control for knock-out and rescue of Dgcr8 and Dicer, are shown at the right of each panel.

https://doi.org/10.7554/eLife.44171.005
Figure 2—figure supplement 1
Viral entry in miRNA-deficient and wild-type ESCs.

To rule out morphological changes affecting the outcome of viral infections, a binding and entry assay was performed. Cells were infected at 4°C which inhibits entry of viral particles, but allows binding of virions. At t = 0, cells are moved to 37°C resulting in a synchronized entry of virus particles allowing an accurate quantification of how much virus enters the cell and virus replication kinetics. Cells were infected with either TMEV (top panels) or Influenza A virus (bottom panels) and viral RNA was quantified by qRT-PCR. For all viruses, binding was comparable in all cell lines. Differences in TMEV kinetics were greater soon after the entry phase and continued to increase towards the end of the experiment. Differences in IAV became most apparent late in the replication phase, possibly because IAV is very proficient in knock down the IFN response in its host cells. Data show the average (n = 3)±s.d, (*) denotes p-value.

https://doi.org/10.7554/eLife.44171.006
Figure 2—figure supplement 2
miRNA-deficient ESCs express IFN in response to dsRNA.

(a) RT-PCR of poly(I:C) activated ESCs. Only Dgcr8 -/- (lane 6, left panel) and Dicer -/- (lane 5 and 6, right panel) express detectable Ifnb1 transcript (top panels). Actb amplification serves as a loading control. (b) Ifnb mRNA quantification by qRT-PCR after increasing amounts of poly(I:C) in Dgcr8-/- and Dicer-/- mESCs. Data show the average (n = 3)±s.d., normalized to mock, (*) p-value<0.05 by t-test. (c) IFN-β ELISA analyses from mock and poly(I:C) stimulated wild-type and miRNA-deficient ESCs. Data show the average (n = 3)±s.d., (*) p-value<0.05 by two-tailed T test (d) TMEV vRNA quantification in wild-type and miRNA-deficient cell lines after mock or Ruxolitinib treatment (JAK1/2 inhibitor). Data show the average (n = 3)±s.d., (*) p value < 0.05 by Student’s t-test, n.s. non-significant (e) TCID50 quantification after TMEV infection of Dgcr8 (left) and Dicer (right) rescued cell lines.

https://doi.org/10.7554/eLife.44171.007
Figure 3 with 1 supplement
MAVS is downregulated by miRNAs in ESCs.

(a, b) Susceptibility of Dgcr8-/-, Dicer-/- and parental cells to TMEV infection after inhibition of IRF3 (BX795) (a) and Nf-κB (BMS345541) (b), normalized to mock-treated cells. (c) Heat map of significantly differentially expressed proteins (p<0.05) in the absence (Dgcr8-/-) or presence (Dgcr8resc) of miRNAs identified by STRING analysis. (d) Western blot analysis of MAVS expression in miRNA-deficient cells (Dgcr8-/- and Dicer-/-, lanes 2 and 5), wild-type counterparts (Dgcr8+/+ and Dicer+/+, lanes 1 and 4) and respective rescued ESCs lines (Dgcr8resc and Dicerresc, lanes 3 and 6). MAVS quantification normalized to Tubulin and relative to wild-type levels is shown at the top of the panel.

https://doi.org/10.7554/eLife.44171.008
Figure 3—source data 1

Source data of mass spectrometry results.

https://doi.org/10.7554/eLife.44171.010
Figure 3—figure supplement 1
miRNAs regulate MAVS and mitochondrial activity.

(a) Treatment of mESCs with inhibitors of IRF3 and NF-κB activation improves viral replication to a much greater extend in the Dgcr8-/- and Dicer-/- cells compared to the parental cells, suggesting that the inhibitory effect that miRNAs have on the IFN response is upstream of these two transcription factors. (b) Western blot analyses of phospho-IRF3 upon stimulation of Dgcr8-/- ESCs with poly(I:C) in the presence or absence of BX759 (inhibitor IRF3 phosphorylation). Tubulin serves as a loading control (c) Quantification of Tnf mRNA upon poly(I:C) stimulation in Dgcr8+/+ and Dgcr8-/- in the presence or absence of BMS345541 (inhibitor of NFkB activation). Data show the average (n = 2) normalized to Gapdh (d) Quantification of oxidative phosphorylation activity by Rhodamine 123 uptake assay in Dgcr8 (left) and Dicer (right) cell lines. (e) qRT-PCR quantification of Mavs mRNA. Mavs expression is higher in the absence of miRNAs (Dgcr8-/- and Dicer-/-), when compared to parental cell lines (Dgcr8+/+ and Dicer+/+). Data show the average (n = 3)±s.d, (*) denotes p<0.05, t-test. (f) Western blot analyses for MDA5, RIG-I and PKR for wild-type cell lines (+/+), Dgcr8 and Dicer KO (-/-), and the respective rescued cell lines (resc). Quantification of the signal normalized to Tubulin and relative to wild-type is shown at the top of each panel.

https://doi.org/10.7554/eLife.44171.009
ESCs regain Ifnb1 expression after MAVS overexpression.

(a) Dual luciferase assay with Mavs, Rig-I and Mda5 3’UTRs in miRNA-deficient cells lines (Dgcr8-/- and Dicer-/-). Data show the average (n = 3)±s.d normalized to Renilla and relative to the parental lines, (*) p-value<0.05 by t-test (b) Western blot of cell line overexpressing MAVS lacking the 3’UTR in Dgcr8+/+ cells (lane 3). MAVS quantification normalized to Tubulin and relative to wild-type is shown at the top (c) Susceptibility (TCID50/ml) of same cells lines as in (b) to TMEV infection (left panel) and quantification of viral RNA after TMEV infections in the same cell lines (right panel). Data show the average (n = 5)±s.d. (*) p-value<0.05 by t-test (d) Ifnb mRNA expression after poly(I:C) transfection of the same cell lines as in (b), average is represented (n = 3)±s.d, normalized to Dgcr8+/+ cell line, (*) p-value<0.05 by t-test.

https://doi.org/10.7554/eLife.44171.011
Figure 5 with 3 supplements
MiR-673–5p regulates MAVS.

(a) Transfection of miRNA mimics miR-125a-5p, miR-125b-5p, miR-185–5p and miR-673–5p in Dgcr8-/- cells followed by MAVS western blot. MAVS quantification normalized to Tubulin and relative to wild-type is shown at the top (b) Quantification of TMEV replication by qRT-PCR in the same cell lines as in (a) (n = 3) (c) Western blot analysis of MAVS expression in Dgcr8+/+ cells transfected with antagomirs against miR-125a-5p, miR-125b-5p and miR-673–5p. MAVS quantification normalized to Tubulin and relative to wild-type is shown at the top (d) Western blot analysis of MAVS expression in miR-673-/- cell lines. MAVS quantification normalized to Tubulin and relative to wild-type is shown at the top (e) Quantification of TMEV vRNA in miR-673 CRISPR knock-out cell lines in a Dgcr8+/+ background. Data show the average (n = 3)±s.d (*) p-value<0.05 by t-test. (f) Quantification of TMEV vRNA shown as fold-change compared to mock upon JAK1/2 inhibition by Ruxolitinib treatment. Data show the average (n = 3)±s.d (g) qRT-PCR quantification of mmu-miR-673–5p expression after retinoic acid (RA) differentiation of ESC. Data show the average (n = 2)±s.e.m, normalized to U6 snRNA, (*) p-val <0.05 by t-test.

https://doi.org/10.7554/eLife.44171.012
Figure 5—figure supplement 1
Characterization of MAVS expression in ESCs and regulation by miRNAs.

(a) Dgcr8-/- cells were transfected with miRNA mimics for 24 hr, followed by RNA extraction and Mavs mRNA quantification by qRT-PCR. Transfection of all miRNA mimics result in (non-significant) decrease of Mavs levels. (b) Dgcr8-/- mESCs were first transfected with mimics, as in (a) followed by TMEV infections 36 hr later, and measurement of the TCID50. All mimics except for mir-185-5p resulted in higher TCID50 values compared to mock transfected cells, suggesting that in mESC cells targeting Mavs with these miRNAs leads to a higher susceptibility to viral infection.

https://doi.org/10.7554/eLife.44171.013
Figure 5—figure supplement 2
Characterization of miR-673-/- mouse embryonic stem cells.

(a) Genomic sequencing of miR-673 locus contained in chromosome 12. In WT, the position of the two miRNAs encoded by the locus, miR-673–5p and miR-673–3p are indicated by yellow boxes. For miR-673-/- clones (1,2 and 3) both sequenced alleles are shown with the indicated deletions induced by CRISPR. (b) Quantification of mmu-miR-673–5p abundance by qRT-PCR. Data show the average (n = 3)±s.d, normalized to U6 snRNA. (n.d, not detected) (c) Quantification of Mavs mRNA levels by RT-qPCR. Data show the average of at least (n = 3)±s.d, normalized to Actb, and relative to Dgcr8 +/+.

https://doi.org/10.7554/eLife.44171.014
Figure 5—figure supplement 3
Characterization of miR-673 -/- mouse ESCs.

(a) TMEV vRNA quantification in wild-type and miRNA-deficient cell lines after mock or Ruxolitinib treatment (JAK1/2 inhibitor). Data show the average (n = 3)±s.d., (*) p value < 0.05 by Student’s t-test, n.s. non-significant (b) Growth curves of ESCs. Data show the average (n = 3)±s.d. (c) No obvious morphological changes were observed between wild-type (Dgcr8+/+) and miR-673-deficient ESCs (d) Quantification of Nanog and Pouf5f1 pluripotent markers upon knocking out miR-673 by CRISPR in comparison to wild-type ESCs, data show the average (n = 3)±s.e.m, (n.s.) non-significant by t-test. NIH3T3 serves as a negative control for pluripotent maker expression (e) Quantification of Nanog and Pouf51 expression after reintroduction of miR-673 mimics and controls for 24 or 48 hr. Data show the average (n = 3)±s.e.m. (n.s.) non-significant by t-test.

https://doi.org/10.7554/eLife.44171.015

Data availability

All processed Mass spectrometry data is provided as Figure 3—source data 1, including LFQ intensity values for each protein detected in each of the samples. All raw data are available from corresponding author upon request.

Additional files

Supplementary file 1

Sequences of the oligonucleotides used in this study.

https://doi.org/10.7554/eLife.44171.016
Transparent reporting form
https://doi.org/10.7554/eLife.44171.017

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