1. Cell Biology
  2. Chromosomes and Gene Expression
Download icon

microRNA-mediated regulation of microRNA machinery controls cell fate decisions

  1. Qiuying Liu
  2. Mariah K Novak
  3. Rachel M Pepin
  4. Taylor Eich
  5. Wenqian Hu  Is a corresponding author
  1. Department of Biochemistry and Molecular Biology, Mayo Clinic, United States
Research Advance
Cite this article as: eLife 2021;10:e72289 doi: 10.7554/eLife.72289
7 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements
Ago2 is the major developmentally regulated Argonaute protein in mouse embryonic stem cells (mESCs).

(A) Western blotting in the wild-type (WT), Flag-Ago1, and Flag-Ago2 mESCs. (B) Colony formation assay for the mESCs. The WT mESCs were cultured under the indicated conditions, and the resultant colonies were fixed and stained for AP (alkaline phosphatase activity). The results represent the means (± SD) of four independent experiments. (C) Western blotting in the WT mESCs cultured under the indicated conditions. (D) Outline of identifying miRNAs that can potentially regulate Ago2. (E) Expression levels of the identified miRNAs from (D) in mESCs. CPM: counts per million reads.

Figure 1—figure supplement 1
Expression of Argonaute proteins in mouse embryonic stem cells (mESCs).
Figure 1—figure supplement 2
Mir182 and Mir183 are associated with Ago2 mRNA in mouse embryonic stem cells (mESCs).
Figure 2 with 1 supplement
Mir182/Mir183 regulate Ago2 and maintain stemness in mouse embryonic stem cells (mESCs).

(A) qRT-PCR on Mir182 and Mir183. For each miRNA, the expression level in wild-type (WT) cells was set as one for relative comparison. U6 RNA was used for normalization. The results represent the means (± SD) of three independent replicates. (B) Western blotting in the WT, Mir182Δ, Mir183Δ, and Mir182Δ/Mir183Δ mESCs. GAPDH was used for normalization in calculating the relative expression levels. (C) Colony formation assay for mESCs. The mESCs were cultured in 15% FBS+ Lif for 7 days, and the resultant colonies were fixed and stained for alkaline phosphatase (AP). (D) Exit pluripotency assay for mESCs. The mESCs were induced to exit pluripotency in medium without Lif for 2 days and then switched to 2i + Lif medium for 5 days. The resultant colonies were fixed and stained for AP. In (C and D), the colony morphology and AP intensity were evaluated through microscopy; 100–200 colonies were examined each time to determine the percentage of undifferentiated colonies. The results represent the means (± SD) of three independent experiments. (E) Western blotting of pluripotency factors during embryoid body (EB) formation.

Figure 2—figure supplement 1
Ago2 mRNA is a target of Mir182 and Mir183 in mouse embryonic stem cells (mESCs).
Figure 3 with 1 supplement
Mir182/Mir183-mediated repression of Ago2 is required for maintaining pluripotency.

(A) Mutating Mir182- and Mir183-binding sites in Ago2 mRNA’s 3’UTR via genome editing. (B) Genotyping of the Ago2 3’UTR mutant. The PCR was performed using the oligos (F and R) indicated in (A). (C) Western blotting in the wild-type (WT), Ago2 3’UTR mutant, Mir182Δ/ Mir183Δ, and Mir182Δ/ Mir183Δ/Ago2 3’UTR mutant. (D) Colony formation assay for mouse embryonic stem cells (mESCs). (E) Exit pluripotency assay for mESCs. In (D and E), the colony morphology and alkaline phosphatase (AP) intensity were evaluated through microscopy. The results represent the means (± SD) of four independent experiments. *p < 0.05 by the Student’s t-test. Western blotting of pluripotency factors in day 5 embryoid bodies (EBs).

Figure 3—figure supplement 1
Inhibition of Mir182/Mir183-mediated regulation of Ago2 in mouse embryonic stem cells (mESCs).
The stemness defects in the 3’UTR mutant mouse embryonic stem cells (mESCs) are caused by elevated let-7 microRNAs (miRNAs).

(A) Relative levels of miRNAs, let-7 pri-miRNAs, and let-7 pre-miRNAs in the wild-type (WT) and the Ago2 3’UTR mutant mESCs. For each miRNA, pri-miRNA, and pre-miRNA, the expression level in WT cells was set as one for relative comparison. U6 RNA was used for normalization in miRNA and pre-miRNA quantification; 18 S rRNA was used for normalization in pri-miRNA quantification. The heatmap was generated from the means of three independent replicates. (B) Colony formation assay for WT and the Ago2 3’UTR mutant mESCs cultured in the presence of 500 nM anti-let-7 locked nucleic acid (LNA) or a control LNA. The results represent three independent experiments. *p < 0.05, and n.s. not significant (p > 0.05) by the Student’s t-test.

Figure 5 with 1 supplement
Mir182/Mir183 and Trim71 function in parallel to repress Ago2 mRNA in mouse embryonic stem cells (mESCs).

(A) Western blotting in the wild-type (WT) mESCs expressing either a vector or FLAG-Trim71 and in the 3’UTR mutant mESCs expressing either a vector or FLAG-Trim71. (B) Western blotting in the WT, 3’UTR mutant, and 3’UTR mutant/CLIPΔ mESCs. In (A and B), GAPDH was used for normalization in calculating the relative expression levels. (C) Colony formation assay for mESCs. (D) Exit pluripotency assay for mESCs.

Figure 5—figure supplement 1
Generation of the CLIPΔ in the 3’UTR mutant mouse embryonic stem cells (mESCs).
Author response image 1
Author response image 2

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Antibody(Mouse monoclonal) anti-FLAG M2Sigma-AldrichCat# F1804WB (1:5000)
Antibody(Mouse monoclonal) anti-GAPDH (6 C5)Santa Cruz BiotechnologyCat# sc-32233WB (1:5000)
Antibody(Rabbit monoclonal) anti-beta-TubulinSelleckchemCat# A5032WB (1:5000)
Antibody(Rabbit monoclonal) anti-Ago1 (D84G10)Cell Signaling TechnologyCat# 5053WB (1:1000)
Antibody(Rabbit monoclonal) anti-Ago2BimakeCat# A5701WB (1:3000)
Antibody(Mouse monoclonal) anti-Oct-4BD Transduction LaboratoriesCat# 611202WB (1:5000)
Antibody(Rabbit monoclonal) anti-Nanog (D2A3)Cell Signaling TechnologyCat# 8822WB (1:3000)
AntibodyGoat Anti-Rabbit IgG (H L)-HRP ConjugateBio-RadCat# 170-6515WB (1:5000)
AntibodyGoat Anti-Mouse IgG (H L)-HRP ConjugateBio-RadCat# 170-6516WB (1:5000)
Chemical compound, drugDMEM/F-12GibcoCat# 12500096
Chemical compound, drugFBSMilliporeCat# ES-009-B
Chemical compound, drugmLIFMilliporeCat# ESG1107
Chemical compound, drugPD0325901APExBioCat# A3013
Chemical compound, drugCHIR99021APExBioCat# A3011
Chemical compound, drugN2MilliporeCat# SCM012
Chemical compound, drugN27MilliporeCat# SCM013
Chemical compound, drugMEM NEAAGibcoCat# 11140–50
Chemical compound, drugPenicillin-StreptomycinGibcoCat# 11548876
Chemical compound, drugL-GlutaminSigma-AldrichCat# G7513
Chemical compound, drugβ-MercaptoethanolSigma-AldrichCat# M3148
Chemical compound, drugAccutaseMilliporeCat# SF006
Chemical compound, drugFugene6PromegaCat# E2691
Chemical compound, drugPuromycinSigma-AldrichCat# P9620
Chemical compound, drugDoxycyclineSigma-AldrichCat# D9891
Chemical compound, drugProtease inhibitorsBimakeCat# B14001
Chemical compound, drugGelatinSigma-AldrichCat# G1890
Chemical compound, drugOne Step-RNA ReagentBio BasicCat# BS410A
Chemical compound, drugDNaseINEBCat# M0303L
Chemical compound, drugSuperScript II Reverse TranscriptaseInvitrogenCat# 18064014
Chemical compound, drugSsoAdvanced Universal SYBR Green SupermixBio-RadCat# 1725270
Chemical compound, drugQ5 High-Fidelity DNA PolymeraseNEBCat# M0491L
Chemical compound, drugControl LNAQiagenCat# 339137
Chemical compound, druganti-let-7 LNAQiagenCat# YFI0450006
Commercial assay or kitAlkaline Phosphatase Assay KitSystem BiosciencesCat# AP100R-1
Commercial assay or kitGibson Assembly Master MixNEBCat# E2611L
Commercial assay or kitPierce BCA Protein Assay KitThermo Fisher ScientificCat# 23225
Commercial assay or kitMir-X miRNA First Strand Synthesis KitTakaraCat# 638313
Cell line (Mus musculus)ES-E14TG2a mESCATCCCRL-1821
Cell line (Mus musculus)FLAG-Ago1 mESCThis paper
Cell line (Mus musculus)FLAG-Ago2 mESCPMID:33599613
Cell line (Mus musculus)Mir182∆ mESCThis paper
Cell line (Mus musculus)Mir183∆ mESCThis paper
Cell line (Mus musculus)Mir182∆/Mir183∆ mESCThis paper
Cell line (Mus musculus)3'UTR Mutant mESCThis paper
Cell line (Mus musculus)Mir182∆/Mir183∆/3'UTR Mutant mESCThis paper
Recombinant DNA reagentPiggyBac-based dox-inducible expression vectorPMID:33599613pWH406
Recombinant DNA reagentInducible GFP expressing vectorPMID:33599613pWH1055
Recombinant DNA reagentInducible mouse Mir182 expressing vectorThis paperpWH1039
Recombinant DNA reagentInducible mouse Mir183 expressing vectorThis paperpWH1040
Recombinant DNA reagentsgRNA and Cas9 expressing vector (pX458) pWH464AddgeneCat# 48138
Recombinant DNA reagentSuper PiggyBac Transposase expressing vector (pWH252)System BiosciencesCat# PB210PA-1

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)