microRNA-mediated regulation of microRNA machinery controls cell fate decisions

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Version of Record published: October 11, 2021 (This version)
Accepted Manuscript published: October 1, 2021 (Go to version)
Accepted: September 30, 2021
Received: July 21, 2021
Preprint posted: July 21, 2021 (Go to version)

1. Builds upon
Repressing Ago2 mRNA translation by Trim71 maintains pluripotency through inhibiting let-7 microRNAs

Qiuying Liu, Xiaoli Chen ... Wenqian Hu

Research Article Feb 25, 2021
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Results
Discussion
Materials and methods
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Abstract

microRNAs associate with Argonaute proteins, forming the microRNA-induced silencing complex (miRISC), to repress target gene expression post-transcriptionally. Although microRNAs are critical regulators in mammalian cell differentiation, our understanding of how microRNA machinery, such as the miRISC, are regulated during development is still limited. We previously showed that repressing the production of one Argonaute protein, Ago2, by Trim71 is important for mouse embryonic stem cells (mESCs) self-renewal (Liu et al., 2021). Here, we show that among the four Argonaute proteins in mammals, Ago2 is the major developmentally regulated Argonaute protein in mESCs. Moreover, in pluripotency, besides the Trim71-mediated regulation of Ago2 (Liu et al., 2021), Mir182/Mir183 also repress Ago2. Specific inhibition of this microRNA-mediated repression results in stemness defects and accelerated differentiation through the let-7 microRNA pathway. These results reveal a microRNA-mediated regulatory circuit on microRNA machinery that is critical to maintaining pluripotency.

Introduction

microRNAs (miRNAs) are endogenous ~22 nucleotide (nt) RNAs with critical roles in modulating gene expression under diverse biological contexts (Bartel, 2009; Bartel, 2018). Most miRNAs are produced from long primary transcripts (pri-miRNAs) through successive processing by two double-stranded RNA (dsRNA) endonucleases named Drosha and Dicer, generating pre-miRNAs and ~22 nt dsRNAs, respectively. One RNA strand in the ~22 nt dsRNA, the mature miRNA, is selectively incorporated into the Argonaute (Ago) protein, forming the miRNA-induced silencing complex (miRISC) (Ha and Kim, 2014). In animals, miRISC recognizes its target mRNAs through partial base pairings mediated by the miRNA (Bartel, 2009). The Ago protein recruits GW182 proteins to down-regulate target mRNA expression through mRNA degradation and/or translational repression (Nilsen, 2007). Although miRNAs play critical regulatory roles in mammalian cell differentiation (Ameres and Zamore, 2013; Ebert and Sharp, 2012), our understanding on how miRNA machinery, particularly the miRISC, are regulated during development is still limited.

We recently found that Ago2, a key component in the miRISC, is repressed at the mRNA translation level by an RNA-binding protein named Trim71 in mouse embryonic stem cells (mESCs) (Liu et al., 2021). This repression of Ago2 inhibits stem cell differentiation mediated by the conserved pro-differentiation let-7 miRNAs (Büssing et al., 2008; Liu et al., 2021). These results suggest that Ago2 is developmentally regulated during stem cell self-renewal and differentiation, and beg for characterization of additional regulators of Ago2. Moreover, besides Ago2, there are three additional Ago proteins (Ago1, Ago3, Ago4) in mammals that function redundantly in the miRNA pathway (Meister, 2013). The relative abundance of these Ago proteins and their contribution to miRNA activities during cell differentiation, however, are still unknown.

Here, using mESC fate decisions between pluripotency and differentiation as a mammalian cell differentiation model, we determined that Ago2 is the predominant Ago protein in mESCs, and Ago2 level increases when mESCs exit pluripotency. In the pluripotent state, Mir182 and Mir183, two conserved miRNAs abundantly expressed in mESCs, repress Ago2 and control the stemness of mESCs. Specific inhibition of Mir182/Mir183-mediated repression of Ago2 results in stemness defects and accelerated differentiation of mESCs through the let-7 miRNA pathway. Collectively, these results reveal an miRNA-mediated regulatory circuit on the miRNA machinery that is critical to maintaining pluripotency.

Results

Ago2 is the predominant developmentally regulated Ago protein in mESCs

Mammals have four Ago proteins (Ago1–4) that function redundantly in miRNA-mediated regulations (Meister, 2013). Transcriptomic profiling on mESCs from different laboratories indicated that mESCs express only Ago1 and Ago2 (Figure 1—figure supplement 1A; Liu et al., 2021; Marks et al., 2012). To examine the relative abundance of Ago1 and Ago2 at the protein level, we generated mESCs with a Flag-tag knocked-in at the N-terminus of the Ago1 and Ago2 loci, respectively, via CRISPR/Cas9-mediated genome editing (Figure 1—figure supplement 1B, C). These mESCs with the Flag-tag knocked-in displayed no stemness defects compared to the wild-type (WT) mESCs (Figure 1—figure supplement 1D) and enabled us to use the same antibody (e.g., anti-Flag) to compare the relative abundance of Ago1 and Ago2. Western blotting via an anti-Flag antibody indicated that Ago2 is the predominant Ago protein in mESCs at the protein level (Figure 1A).

Figure 1 with 2 supplements see all

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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—source data 1 Tiff files of raw gel images for Figure 1A and C; Figure 1—figure supplement 1C. Excel files of numbers for Figure 1B and E; Figure 1—figure supplement 1A, D, Figure 1—figure supplement 2.: https://cdn.elifesciences.org/articles/72289/elife-72289-fig1-data1-v2.zip
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To examine whether Ago2 level is regulated during mESCs differentiation, we cultured mESCs under three different conditions that mimic three different developmental stages: ground/naive state (in 2i + Lif), primed state (in 15% FBS+ Lif), and differentiating state (in 15% FBS without Lif), which resulted in decreasing stemness in mESCs, as determined by the colony formation assay (Figure 1B). Western blotting indicated that Ago2 level increased when mESCs exited pluripotency (Figure 1C). This result indicated that Ago2 is developmentally regulated in mESCs, and Ago2 level is repressed in the pluripotent state.

Mir182/Mir183 regulate Ago2 and maintain stemness in mESCs

To determine how Ago2 is regulated in mESCs, we hypothesized that miRNAs expressed in mESCs might contribute to the repression of Ago2 because miRNAs are important negative regulators of gene expression. We identified the conserved miRNA-binding sites in the 3’UTR of Ago2 mRNA through TargetScan (Agarwal et al., 2015) and then examined the expression level of the corresponding miRNAs in mESCs using existing small-RNA-seq datasets (Liu et al., 2021 Figure 1D). This analysis revealed that among the miRNAs that can potentially regulate Ago2, Mir182, and Mir183, two miRNAs from the same miRNA family that are abundantly expressed in stem cells Dambal et al., 2015, have significantly higher expression levels (Figure 1E). Interestingly, Mir182/Mir183 decrease when mESCs transition from the ground state to the primed and differentiating state (Hadjimichael et al., 2016; Wang et al., 2017), which negatively correlates with the Ago2 expression pattern during this transition (Figure 1C). These observations suggest that Ago2 is repressed by Mir182/Mir183 in mESCs. Consistent with this notion, using RNA antisense purification, we found that Mir182 and Mir183 specifically associated with Ago2 mRNA in mESCs (Figure 1—figure supplement 2).

Two lines of evidence indicated that Mir182/Mir183 regulate Ago2 mRNA. First, Ago2 increased when Mir182, Mir183, or both Mir182 and Mir183 were knocked out in mESCs (Figure 2—figure supplement 1A, Figure 2A and B). Second, when either Mir182 or Mir183 was over-expressed in the WT mESCs (Figure 2—figure supplement 1B), the Ago2 level decreased (Figure 2—figure supplement 1C). The results from these loss-of-function and gain-of-function experiments argue that Mir182/Mir183 repress Ago2 expression in mESCs.

Figure 2 with 1 supplement see all

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*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—source data 1 Tiff files of raw gel images for Figure 2B and E; Figure 2—figure supplement 1C. Excel files of numbers for Figure 2A, C and D; Figure 2—figure supplement 1B.: https://cdn.elifesciences.org/articles/72289/elife-72289-fig2-data1-v2.zip
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Interestingly, Mir182Δ, Mir183Δ, and Mir182Δ/Mir183Δ mESCs displayed defects in self-renewal (Figure 2C), as determined by the colony formation assay in the 15% FBS + Lif medium, where differentiation was not blocked by the two inhibitors in the 2i + Lif medium. Moreover, these miRNA knockout mESCs had accelerated differentiation, as revealed by the exit pluripotency assay (Figure 2D), which evaluates the rate ESCs exit the pluripotent state (Betschinger et al., 2013), and by the measurement of pluripotency factors through Western blotting on differentiating embryonic bodies (Figure 2E). These cellular phenotypes suggest that Mir182/Mir183-mediated regulation of Ago2 is important to mESCs.

Mir182/Mir183-mediated repression of Ago2 is required for maintaining pluripotency

A caveat in interpreting results from miRNA knockout and over-expression experiments is the pleiotropic effects. Because each miRNA can regulate hundreds of mRNAs, when an miRNA is knocked out or over-expressed, hundreds of miRNA:mRNA interactions are altered, making it difficult to determine whether a specific miRNA:mRNA interaction contributes to the phenotypical changes.

To address this issue and specifically examine the functional significance of Mir182/Mir183-mediated regulation of Ago2 in mESCs, we mutated the Mir182/Mir183-binding sites in the 3’UTR of Ago2 mRNA via CRISPR/Cas9-mediated genome editing (Figure 3A and B). Two observations indicated that the mutations disrupted the interaction between Ago2 mRNA and Mir182/Mir183. First, similar to the miRNA knockout mESCs (Figure 2B), Ago2 increased in the 3’UTR mutant mESCs (Figure 3C). Second, in contrast to the results in the WT mESCs (Figure 2—figure supplement 1C), over-expression of either Mir182 or Mir183 in the 3’UTR mutant mESCs did not decrease Ago2 (Figure 3—figure supplement 1A, B). Notably, in the Mir182Δ/Mir183Δ mESCs, these mutations did not increase Ago2 (Figure 3C), indicating the increased Ago2 from these mutations in the WT mESCs is dependent on Mir182/Mir183. Moreover, the 3’UTR mutations did not significantly alter the Mir182/Mir183 levels in mESCs (Figure 3—figure supplement 1C). Altogether, these observations indicated that the functional significance of Mir182/Mir183-mediated repression of Ago2 could be specifically evaluated in the 3’UTR mutant mESCs.

Figure 3 with 1 supplement see all

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*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—source data 1 Tiff files of raw gel images for Figure 3C and F; Figure 3—figure supplement 1B. Excel files of numbers for Figure 3D and E; Figure 3—figure supplement 1A, C.: https://cdn.elifesciences.org/articles/72289/elife-72289-fig3-data1-v2.zip
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When subject to the colony formation assay, the 3’UTR mutant mESCs displayed a defect in maintaining undifferentiated colonies (Figure 3D), indicating compromised self-renewal. When differentiation was evaluated by the exit pluripotency assay, the 3’UTR mutant mESCs had an increased differentiation rate (Figure 3E). Consistent with these findings, differentiating embryonic bodies from the 3’UTR mutant mESCs had a lower amount of pluripotency factors (Figure 3F). Collectively, these results indicate that Mir182/Mir183-mediated repression of Ago2 is important for mESC self-renewal and proper differentiation.

Mir182/Mir183-mediated repression of Ago2 in mESCs inhibits the let-7 miRNA-mediated differentiation pathway

Two observations lead us to the hypothesis that Mir182/Mir183-mediated repression of Ago2 in mESCs counteracts the differentiation pathway controlled by the let-7 miRNAs, a conserved miRNA family that promotes stem cell differentiation (Roush and Slack, 2008). First, in Dgcr8Δ mESCs, where endogenous miRNAs’ biogenesis is blocked, ectopic expression of Mir183 inhibits the stem cell differentiation triggered by exogenous let-7 miRNA (Wang et al., 2017). Second, our recent study indicated that increasing Ago2 levels in mESCs results in stemness defects in a let-7-miRNA-dependent manner. This specificity on let-7 miRNAs is because the pro-differentiation let-7 miRNAs are actively transcribed in mESCs, and the increased Ago2 binds and stabilizes the let-7 miRNAs that are otherwise degraded in mESCs, thereby promoting mESCs differentiation (Liu et al., 2021).

To test this hypothesis, we examined the expression of let-7 miRNAs. The 3’UTR mutant mESCs had significantly higher let-7 miRNAs than the WT mESCs (Figure 4A). This increase is specific to let-7 miRNAs because non-let-7 miRNAs were not elevated (Figure 4A). Moreover, consistent with our previous observation that increased Ago2 stabilizes mature let-7 miRNAs (Liu et al., 2021), the pri-let-7 miRNAs and the pre-let-7 miRNAs were not significantly increased in the 3’UTR mutant mESCs (Figure 4A). To determine whether the increased let-7 miRNAs are responsible for the stemness defects in the 3’UTR mutant mESCs, we inhibited let-7 miRNAs using locked nucleic acid (LNA) antisense oligonucleotides targeting the conserved seed sequence of let-7 miRNAs. When let-7 miRNAs were inhibited, the stemness defects of the 3’UTR mutant mESCs were abolished (Figure 4B), indicating that disruption of Mir182/Mir183-mediated repression of Ago2 in mESCs activates differentiation through the let-7 miRNA pathway.

Figure 4

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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 4—source data 1 Excel files of numbers for Figure 4A and B.: https://cdn.elifesciences.org/articles/72289/elife-72289-fig4-data1-v2.zip
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Mir182/Mir183 and trim71 function in parallel to repress Ago2 mRNA in mESCs

Our previous study indicated that Ago2 mRNA is also repressed by Trim71 in mESCs (Liu et al., 2021). Interestingly, the Trim71-binding site in the 3’UTR of Ago2 mRNA is different from the Mir182/Mir183-binding sites, suggesting that Mir182/Mir183 and Trim71 function in parallel to repress Ago2 mRNA in mESCs. We performed the following experiments to test this.

At the molecular level, we observed that over-expression of Trim71 still repressed Ago2 in the 3’UTR mutant mESCs (Figure 5A), where Mir182/Mir183-mediated repression is abolished (Figure 3). Moreover, in the 3’UTR mutant mESCs, inhibiting Trim71-mediated repression of Ago2 through deleting the Trim71-binding site in the 3’UTR of Ago2 mRNA (CLIPΔ) (Liu et al., 2021) further increased Ago2 level (Figure 5B, Figure 5—figure supplement 1). These results indicate that Trim71 and Mir182/Mir183 independently repress Ago2 mRNA in mESCs.

Figure 5 with 1 supplement see all

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*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—source data 1 Tiff files of raw gel images for Figure 5A and B; Figure 5—figure supplement 1. An Excel file of numbers for Figure 5C and D.: https://cdn.elifesciences.org/articles/72289/elife-72289-fig5-data1-v2.zip
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At the cell function level, we found that introducing the CLIPΔ in the 3’UTR mutant mESCs further decreased stem cell self-renewal, as determined by the colony formation assay (Figure 5C), and accelerated differentiation, as measured by the exit pluripotency assay (Figure 5D). These observations argue that Trim71 and Mir182/Mir183 function independently in regulating stemness in mESCs through modulating Ago2 mRNA.

Collectively, these findings indicate that Mir182/Mir183 and Trim71 function in parallel to repress Ago2 mRNA in mESCs.

Discussion

Our data reveal that the predominant Ago protein in mESCs, Ago2, is developmentally regulated, with gradually increasing levels when mESCs exit pluripotency. Two miRNAs abundantly expressed in mESCs, Mir182/Mir183, contribute to the repression of Ago2 in the pluripotent state. This miRNA-mediated regulation of Ago2 is critical to maintaining stemness. Our findings raise several interesting aspects of miRNAs in stem cell biology.

First, since Ago2 is the predominant Ago protein in mESCs, the Ago2 expression pattern during mESCs’ transition from self-renewal to differentiation argues that although certain individual miRNAs may be required for pluripotency (e.g., Mir182/Mir183), the global miRNA activity is suppressed in the pluripotent state and induced when mESCs initiate differentiation. Consistent with this notion, knocking out key components in global miRNA biogenesis, such as Dgcr8 (Wang et al., 2007), Dicer (Kanellopoulou et al., 2005; Murchison et al., 2005), or Ago2 in the miRISC (Liu et al., 2021), does not negatively affect mESCs self-renewal. However, differentiation in all these mutant mESCs is severely compromised. Thus, at the global level, miRNAs may play more important roles in mESC differentiation.

Second, previous studies indicate that the two components of the miRISC, the Ago protein and its associated miRNA, mutually regulate each other. In the absence of miRNAs, the Ago protein is destabilized (Martinez and Gregory, 2013; Smibert et al., 2013), while miRNAs are also unstable if they are not associated with Ago proteins (Winter and Diederichs, 2011). Thus, the effective miRNA activity depends on the limiting component in the miRISC. Our previous studies indicated that the conserved pro-differentiation let-7 miRNAs are sensitive to Ago2 levels because an increase of Ago2 results in specific stabilization of let-7 miRNAs that are otherwise degraded (Liu et al., 2021). Thus, for let-7 miRISC, Ago2 is possibly the limiting component in mESCs. Repression of Ago2 by either Mir182/Mir183 as we characterized here or Trim71 as we identified previously (Liu et al., 2021) likely limits the effective let-7 miRISCs. Interestingly, the pro-differentiation let-7 miRICSs can positively auto-regulate their own biogenesis through inhibiting Lin28a, a conserved let-7 target, because Lin28a inhibits the biogenesis of let-7 miRNAs through promoting their pre-miRNA degradation (Tsialikas and Romer-Seibert, 2015). Thus, the effective let-7 miRNAs need to be tightly controlled in stem cells. The two repression mechanisms on Ago2 mRNA contribute to limiting the amount of effective let-7 miRISCs and maintaining pluripotency in mESCs. We speculate that similar mechanisms of regulating miRISCs by RNA-binding proteins and miRNAs may exist in other developmental processes. Moreover, Ago2 is dysregulated under many pathological conditions, such as cancer (Adams et al., 2014). Thus, regulating miRISCs through modulating Ago2 levels may also contribute to pathogenesis.

Finally, it is noticed that the Mir182Δ/Mir183Δ mESCs displayed stronger defects in self-renewal and differentiation than the 3’UTR mutant mESCs did (Figure 2C and D versus Figure 3D and E). Thus, besides Ago2 mRNA, Mir182/Mir183 may regulate additional mRNAs that are important for stem cell biology.

Materials and methods

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Antibody	(Mouse monoclonal) anti-FLAG M2	Sigma-Aldrich	Cat# F1804	WB (1:5000)
Antibody	(Mouse monoclonal) anti-GAPDH (6 C5)	Santa Cruz Biotechnology	Cat# sc-32233	WB (1:5000)
Antibody	(Rabbit monoclonal) anti-beta-Tubulin	Selleckchem	Cat# A5032	WB (1:5000)
Antibody	(Rabbit monoclonal) anti-Ago1 (D84G10)	Cell Signaling Technology	Cat# 5053	WB (1:1000)
Antibody	(Rabbit monoclonal) anti-Ago2	Bimake	Cat# A5701	WB (1:3000)
Antibody	(Mouse monoclonal) anti-Oct-4	BD Transduction Laboratories	Cat# 611202	WB (1:5000)
Antibody	(Rabbit monoclonal) anti-Nanog (D2A3)	Cell Signaling Technology	Cat# 8822	WB (1:3000)
Antibody	Goat Anti-Rabbit IgG (H L)-HRP Conjugate	Bio-Rad	Cat# 170-6515	WB (1:5000)
Antibody	Goat Anti-Mouse IgG (H L)-HRP Conjugate	Bio-Rad	Cat# 170-6516	WB (1:5000)
Chemical compound, drug	DMEM/F-12	Gibco	Cat# 12500096
Chemical compound, drug	FBS	Millipore	Cat# ES-009-B
Chemical compound, drug	mLIF	Millipore	Cat# ESG1107
Chemical compound, drug	PD0325901	APExBio	Cat# A3013
Chemical compound, drug	CHIR99021	APExBio	Cat# A3011
Chemical compound, drug	N2	Millipore	Cat# SCM012
Chemical compound, drug	N27	Millipore	Cat# SCM013
Chemical compound, drug	MEM NEAA	Gibco	Cat# 11140–50
Chemical compound, drug	Penicillin-Streptomycin	Gibco	Cat# 11548876
Chemical compound, drug	L-Glutamin	Sigma-Aldrich	Cat# G7513
Chemical compound, drug	β-Mercaptoethanol	Sigma-Aldrich	Cat# M3148
Chemical compound, drug	Accutase	Millipore	Cat# SF006
Chemical compound, drug	Fugene6	Promega	Cat# E2691
Chemical compound, drug	Puromycin	Sigma-Aldrich	Cat# P9620
Chemical compound, drug	Doxycycline	Sigma-Aldrich	Cat# D9891
Chemical compound, drug	Protease inhibitors	Bimake	Cat# B14001
Chemical compound, drug	Gelatin	Sigma-Aldrich	Cat# G1890
Chemical compound, drug	One Step-RNA Reagent	Bio Basic	Cat# BS410A
Chemical compound, drug	DNaseI	NEB	Cat# M0303L
Chemical compound, drug	SuperScript II Reverse Transcriptase	Invitrogen	Cat# 18064014
Chemical compound, drug	SsoAdvanced Universal SYBR Green Supermix	Bio-Rad	Cat# 1725270
Chemical compound, drug	Q5 High-Fidelity DNA Polymerase	NEB	Cat# M0491L
Chemical compound, drug	Control LNA	Qiagen	Cat# 339137
Chemical compound, drug	anti-let-7 LNA	Qiagen	Cat# YFI0450006
Commercial assay or kit	Alkaline Phosphatase Assay Kit	System Biosciences	Cat# AP100R-1
Commercial assay or kit	Gibson Assembly Master Mix	NEB	Cat# E2611L
Commercial assay or kit	Pierce BCA Protein Assay Kit	Thermo Fisher Scientific	Cat# 23225
Commercial assay or kit	Mir-X miRNA First Strand Synthesis Kit	Takara	Cat# 638313
Cell line (Mus musculus)	ES-E14TG2a mESC	ATCC	CRL-1821
Cell line (Mus musculus)	FLAG-Ago1 mESC	This paper
Cell line (Mus musculus)	FLAG-Ago2 mESC	PMID:33599613
Cell line (Mus musculus)	Mir182∆ mESC	This paper
Cell line (Mus musculus)	Mir183∆ mESC	This paper
Cell line (Mus musculus)	Mir182∆/Mir183∆ mESC	This paper
Cell line (Mus musculus)	3'UTR Mutant mESC	This paper
Cell line (Mus musculus)	Mir182∆/Mir183∆/3'UTR Mutant mESC	This paper
Recombinant DNA reagent	PiggyBac-based dox-inducible expression vector	PMID:33599613	pWH406
Recombinant DNA reagent	Inducible GFP expressing vector	PMID:33599613	pWH1055
Recombinant DNA reagent	Inducible mouse Mir182 expressing vector	This paper	pWH1039
Recombinant DNA reagent	Inducible mouse Mir183 expressing vector	This paper	pWH1040
Recombinant DNA reagent	sgRNA and Cas9 expressing vector (pX458) pWH464	Addgene	Cat# 48138
Recombinant DNA reagent	Super PiggyBac Transposase expressing vector (pWH252)	System Biosciences	Cat# PB210PA-1

All the antibodies, plasmids, and oligonucleotides used in this study are listed in Supplementary file 1.

Cell lines

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All the cell lines from this study are based on ES-E14TG2a mESC (ATCC, CRL-1821). They are listed in Supplementary file 1. The ES-E14TG2a mESCs were authenticated through STR profiling and were negative for mycoplasma contamination determined by a PCR-based kit.

CRISPR/Cas9-mediated genome editing in mESCs

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To generate the FLAG-Ago1, FLAG-Ago2 mESCs, or Ago2 3’UTR mutant mESCs, cells were co-transfected with 2 µg of pWH464 (pSpCas9(BB)-2A-GFP [pX458]) expressing the corresponding targeting sgRNA and 1 µg of the corresponding donor oligo or plasmid using the Fugene6 (Promega). To generate Mir182Δ and Mir183Δ mESCs, cells were transfected with 2 µg of pWH464 expressing a pair of sgRNAs targeting pri-Mir182 or pri-Mir183. The transfected cells were subject to single cell sorting and the resulting clones were subject to genotyping to identify the correct clones.

qRT-PCR

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For pri-miRNA quantification, reverse transcription was performed using random hexamers and Superscript II Reverse Transcriptase. Pre-miRNA and miRNA quantifications were using the Takara’s Mir-X miRNA quantification method. qPCR was performed in triplicate for each sample using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) on a CFX96 real-time PCR detection system (Bio-Rad).

Western blotting

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Proteins were harvested in RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail) and quantified with a BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein samples were resolved by SDS-PAGE, and then transferred to PVDF membranes. Western blotting was performed using a BlotCycler (Precision Biosystems) with the corresponding primary and secondary antibodies. The membranes were then treated with the Western ECL substrate (Bio-Rad), and the resulting signal was detected using an ImageQuant LAS 500 instrument (GE Healthcare).

Colony formation assay and exit pluripotency assay

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For colony formation assay, 500 cells were plated on a 12-well plate in 2i + Lif media or Lif media (DMEM/F12 supplemented with 15% FBS, 1× penicillin/streptomycin, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, and 1000 U/ml Lif). For exit from pluripotency assay, 1000 cells were plated on a gelatin-coated six-well plate in differentiation media (DMEM/F12 supplemented with 15% FBS, 1× penicillin/streptomycin, 0.1 mM non-essential amino acids, 2 mM L-glutamine, and 0.1 mM 2-mercaptoethanol) for 2 days, then cultured in 2i + Lif media for another 5 days. Colonies were stained using AP staining kit and grouped by differentiation status 6–7 days after plating.

Embryoid body formation

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For differentiation via embryoid body (EB) formation, 3 × 10⁶ cells were plated per 10 cm bacterial grade Petri dish and maintained on a horizontal rotator with a rotating speed of 30 rpm in differentiation media. The resultant EBs were harvested at the indicated time points.

RNA antisense purification mESCs were crosslinked with 0.1% formaldehyde for 5 min at room temperature, and the crosslinking reaction was quenched by adding 1/20 volume of 2.5 M glycine and incubating the mESCs at room temperature for 10 min on a rotating platform. The cells were then harvested and lysed in cell lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Tween-20, with freshly added proteinase inhibitors). The cell lysate was cleared by centrifugation at 20,000 g for 10 min at 4°C. The resulting supernatant was used for RNA antisense purification; 5 mg lysate in 500 µl lysis buffer was used for each purification. Specifically, a set of 5’-end biotinylated anti-sense DNA oligos and 5 µl RNase inhibitor (NEB) were added to the lysate, resulting in a final concentration of 0.1 µM for each oligo. The lysate was incubated at room temperature for 1 hr on a rotating platform. Then, 100 µl Dynabeads MyOne Streptavidin C1 (Invitrogen) was added and the lysate further incubated for 30 min at room temperature on a rotating platform. The magnetic beads were isolated through a magnetic stand and then subject to four washes, with each wash in 500 µl high salt wash buffer (5× PBS, 0.5% sodium deoxycholate, 1% Triton X-100). The washed beads were resuspended in 100 µl DNaseI digestion mix (1× DNaseI digestion buffer with 5 µl DNaseI [NEB]) and incubated at 37°C for 20 min, followed by adding 350 µl LET-SDS buffer (25 mM Tris-HCl pH 8.0, 100 mM LiCl, 20 mM EDTA pH 8.0, 1% SDS) and 50 µl proteinase K (20 mg/ml, Thermo Fisher Scientific). The beads were then incubated on a thermomixer at 55°C 1000 rpm for 2 hr. The RNA was isolated through phenol extraction and isopropanol precipitation with glycoblue (Ambion) as a coprecipitant.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1–5.

The following previously published data sets were used

1. Marks H
2. Menafra R
3. Kalkan T
4. Denissov S
5. Jones K
6. Hofemeister H
7. Nichols J
8. Kranz A
9. Stewart AF
10. Smith A
11. Stunnenberg HG
(2012) NCBI Gene Expression Omnibus
ID GSE23943. Epigenome and transcriptome of naive pluripotent mouse embryonic stem (ES) cells cultured in 2i serum-free medium.

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE23943
1. Hu W
2. Liu Q
3. Zhang H
4. Chen X
5. Zhang S
(2021) NCBI Gene Expression Omnibus
ID GSE138284. Studies on Trim71 in mouse embryonic stem cells.

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE138284

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Decision letter

Timothy W Nilsen

Reviewing Editor; Case Western Reserve University, United States
James L Manley

Senior Editor; Columbia University, United States
Ravi K Patel

Reviewer; UCSF, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

In aggregate, the data strongly supports the conclusions made. This revised manuscript includes additional data and clarifications: the authors have responded effectively to comments raised in initial review.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting the paper "microRNA-mediated regulation of microRNA machinery controls cell fate decisions" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor.

We are sorry to say that, after consultation with the reviewers, we have decided that this work cannot be considered further for publication by eLife.

The agreement was that the submission was not suitable as a Research Advance. One reviewer judged the results to be a relatively small advance over your earlier paper. The other reviewer commented that a significant number of experiments were required before this could be considered for publication in eLife. Please see the detailed comments of the reviewers below.

Reviewer #1:

The study by Liu and colleagues investigated the molecular mechanisms that regulate the function of Argonaute 2 (AGO2), an essential component of the RNA-induced silencing complex, to control the developmental progression of pluripotent mouse embryonic stem cells (mESCs). Through a series of in vitro molecular assays, the authors showed that AGO2 is the major developmentally regulated Argonaute protein in mESCs, and that AGO2 is repressed by microRNA-182/microRNA-183. The experiments appear well-designed and the methods are technically sound. However, the study provides only marginal conceptual advances, especially given the very recent publication of a study by the same group showing how AGO2 is regulated by the heterochronic gene Trim71 (Liu et al., 2021, eLife). I have two major concerns for the authors to consider:

First, is there a way for the authors to confirm direct binding of microRNA-182/microRNA-183 to Ago2 RNA? If so, I consider this an essential experiment for the authors to carry out, given that the evidence thus far appears indirect (in sillico analysis showing presence of binding motifs and altered expression of Ago2 when the putative binding sites were mutated in cell lines).

Second, the authors have already shown in a previous work that Trim71 also represses Ago2. It is thus surprising to me that in the current study, the authors make no attempt to experimentally link how microRNA-182/microRNA-183 may be working together with Trim71 to regulate Ago2. What is the significance to the overall field to show identify and show additional repressors of Ago2, and how do these newly identified repressors cooperate with Trim71? It is essential for the authors to better link their current work in the context of previous findings by demonstrating how Ago2 repressors may be working together, and whether they may specific functions that distinguish them from each other.

From an experimental standpoint, the authors need to provide better mechanistic insights that explain how microRNA-182/microRNA-183 may cooperate with Trim71 to regulate Ago2 function in mESCs. Otherwise, the conceptual advance provided by the current manuscript appears minor.

Reviewer #2:

Liu et al. investigated the role of micorRNA(miRNA)-mediated regulation of miRNA-induced silencing complex (miRISC) during the differentiation of mouse embryonic stem cells (mESCs). Corroborating previous evidences establishing high mRNA levels for only one paralog of Argonautes (Ago2) in mESCs, the authors demonstrated that the Ago2 is expressed at high levels in mESCs at protein levels too, and that the AGO2 levels are elevated upon mESC differentiation. Using many independent and orthogonal experiments, the authors find with abundance of evidence that miRNAs, miR-182 and miR-183 whose conserved bindings are present in the 3'UTR of Ago2 and whose expression negatively correlates with AGO2 levels in mESCs directly repress AGO2 levels and that the loss of this miRNA-mediated repression of AGO2 promotes enhanced differentiation of mESCs in vitro. The strongest evidence of this direct regulation of AGO2 comes from the experiments where miR-182/183 sites are deleted in the AGO2 3'UTR (3'UTR mutant), which recapitulates the phenotype. While these results robustly show that AGO2/miR-182/183 axis enhances mESC differentiation during the induced transition of mESCs from the ground state to the primed state, this work doesn't show whether this axis is sufficient to control the differentiation in the ground/naive state. Finally, in concordance with their prior findings (Liu et al. 2021), the authors show that the AGO2 levels modulated by miR-182/183 also control the let-7-dependent differentiation of mESCs.

While the previous reports showed that miR-182/183 suppresses differentiation-inducing miRNAs, including let-7, miR-26 and miR-218, this work forms a unique niche and provides a link between miR-182/183 and let-7 in the form of miR-182/183-mediated repression of AGO2. The experiments in this paper follow a logical progression and provide high quality data. The conclusions are mostly supported by the data.

However, in its current form, this paper needs clarification on some fundamental aspects and some additional data. See below for the details.

1) The authors use as readout the reduction in colony formation during the transition of mESCs to the primed state, where the undifferentiated colonies are already reduced to 30%, and where there could be many confounding effects from large transcriptional changes. It is surprising that the authors didn't use the colony formation in naive/ground state as the readout and show that the increased AGO2 levels cause cells to lose stemness. They could potentially see much larger effects in the ground state, attributable directly to AGO2/miR-182/183. If using naive/ground state in these experiments is experimentally not feasible, the authors should discuss that.

2) Though the authors conclude that the elevated levels of AGO2 selectively stabilizes let-7 family members, the mechanism of this selectively is lacking. This model might be an over-simplification, since the increased AGO2 levels can cause system-wide cascade of direct and indirect effects, some of which might be responsible for this increased selectivity. It is likely that LIN28A, which is expressed at higher levels in mESCs and is involved in the well-established feedback loop with let-7 miRNA family, is involved in this let-7-specificity. A plausible alternative hypothesis is that the increased levels of AGO2 in 3'UTR mutant or during differentiation stabilizes all actively produced mature miRNAs equally. However, most miRNAs may not cause an immediate impact on levels of other miRNAs or the miRNA machinery, but the newly formed let-7-miRISCs reduce the high levels of LIN28A, promoting the elevated levels of mature let-7 miRNAs, which competes with other mature miRNAs and saturates the newly formed AGO2 molecules. This hypothesis doesn't assume that the increased AGO2 selectively stabilizes let-7 solitarily.

3) The authors claim that AGO2 repression via miR-182/183 is important for proper differentiation of mESC, however, the data in this paper doesn't show an impact of AGO2 repression on defective differentiation.

Some potential outstanding questions that the authors may want to explore towards this paper or the future work are: (1) how do miR-182/183 levels are modulated during the differentiation process and if modulating their levels is sufficient to promote differentiation in the ground/naive state; (2) this paper suggests that the inhibition of many differentiation-inducing miRNAs that has been shown previously to be trigged by miR-182/183 (Wang et al., 2017), is orchestrated through miR-182/183 mediated suppression of miRISC machinery. It would be of great importance to investigate how do miR-182/183 themselves escape the effect of inhibited miRISC machinery, e.g., do these miRNAs have higher half-life in mESCs? (3) Does the loss of AGO2 repression only enhance the differentiation or does it also trigger defective differentiation? (4) What is the significance of this regulation in vivo.

"Two lines of evidence indicated that miR-182/miR-183 targets Ago2 mRNA." – should use "regulates" instead of "targets", since the evidence for direct targeting comes later in the paper.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "microRNA-mediated regulation of microRNA machinery controls cell fate decisions" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Timothy Nilsen as the Reviewing Editor and James Manley as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Ravi K Patel (Reviewer #1).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

The reviewers and the reviewing editor found the work to be an interesting extension of your previous findings. Nevertheless, all of the reviewers have made relatively minor suggestions for improvement. In particular, as noted by reviewers 1 and 3, it is important to temper your conclusion regarding the role of let-7. Please address these issues as thoroughly as possible before resubmitting.

Reviewer #1:

In this research advance article, Liu et al. investigated the role of micorRNA(miRNA)-mediated regulation of miRNA-induced silencing complex (miRISC) during the differentiation of mouse embryonic stem cells (mESCs). The authors demonstrated that the Ago2 is expressed at high levels in mESCs at protein levels and that the AGO2 levels are elevated upon mESC differentiation. Using multiple independent and orthogonal experiments, the authors find that miRNAs, miR-182 and miR-183 whose conserved binding sites are present in the 3'UTR of Ago2 and whose expression negatively correlates with AGO2 levels in mESCs directly repress AGO2 levels and that the loss of this miRNA-mediated repression of AGO2 promotes enhanced differentiation of mESCs in vitro. The strongest evidence of this direct regulation of AGO2 comes from the experiments where miR-182/183 sites are deleted in the AGO2 3'UTR (3'UTR mutant), which recapitulates the phenotype. These results robustly show that AGO2/miR-182/183 axis enhances mESC differentiation. Finally, in concordance with their prior findings (Liu et al. 2021), the authors show that the AGO2 levels modulated by miR-182/183 or Trim71 control the let-7-dependent differentiation of mESCs. The experiments in this manuscript follow a logical progression and provide high quality data. The conclusions are supported by the data.

Reviewer #2:

In aggregate, the data supports strongly the conclusions made. This revised manuscript includes additional data and clarifications: the authors have responded effectively to comments raised in review.

Reviewer #3:

In this manuscript Liu and colleagues follow-up on their previous work showing that Ago2 expression is regulated in mESC by Trim-71 by proposing that Ago2 expression is also directly regulated by miR-182/miR-183 and that loss of this interaction affect mESC differentiation by resulting in let-7 stabilization.

Using elegant genetic and biochemical approaches they convincingly show that Ago2 is directly repressed by miR-183/miR-183, as either loss of these two miRNAs or mutation of their binding sites in the Ago2 3'UTR results in upregulation of Ago2 expression by approximately 2 fold.

They also show that targeted inactivation of miR-182/183 and, to a slightly lesser extent, mutation of miR-182/miR-183 binding sites in Ago2's 3'UTR promote differentiation of mESC in vitro.

The manuscript is very well written, the experiments are elegant and include all the appropriate controls, and their results are largely consistent with the model proposed by the authors. While the authors convincingly demonstrate that miR-182/182 directly regulate Ago2, it is slightly less clear whether the proposed increased activity of let-7 can fully explain the observed phenotype.

Overall this is a solid manuscript, although as discussed below not all conclusions are supported by conclusive evidence. I concur with reviewer 1 that the study provides relatively minor conceptual advance, but it can be argued that these results will be of interest to specialists in the field of miRNa and ESC biology.

A major concern I have regards the strength of the conclusion that the impaired stem cell function upon loss of miR-182/183 activity on Ago2 is mediated by increased activity of let-7. The LNA-based experiments are suggestive, but although they prove that blocking let-7 helps preventing differentiation, they do not prove that increased let-7 function underlies the observed phenotype in the Ago2 UTR mutant. In fact, the only evidence that let-7 activity is increased is a modest upregulation of let-7 members expression. Whether this is sufficient to affect gene expression to a detectable extent is unclear. The evidence would be strengthened substantially if the authors showed increased let-7-mediated gene repression in the UTR mutant. A straightforward and relatively inexpensive experiment would be to perform RNAseq analysis on the wt and UTR-mutant mESCs. The authors' model leads to the prediction that the a relatively selective de-repression of predicted let-7 targets should be observed in the UTR mutant samples.

1) Figures 2C and 2D appear swapped in their description in the main text (lines 107-114)

2) lines 132-135: I am not sure the authors can define the effect of the 3'UTR as not "in cis". Even if mediated by loss of miRNA binding, as is likely, the effect would still be on the transcript harboring the mutation, so technically, as far as I know, in cis. Perhaps a different language would be more appropriate. For example, the authors could say that the fact that deletion of miR-182/183 in a the 3'UTR-mut background doesn't result in additional upregulation of Ago2 provides additional evidence that the UTR mutation results in Ago2 upregulation by preventing miR-182/183 binding.

3) It appears to me that loss of miR-182/183 has a stronger effect on ESC differentiation than mutation of their Ago2 3'UTR binding sites. This suggests that miR-182 promote ESC pluripotency by additional mechanism independent from Ago2. Perhaps the authors should comment on this in the discussion.

https://doi.org/10.7554/eLife.72289.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1:

The study by Liu and colleagues investigated the molecular mechanisms that regulate the function of Argonaute 2 (AGO2), an essential component of the RNA-induced silencing complex, to control the developmental progression of pluripotent mouse embryonic stem cells (mESCs). Through a series of in vitro molecular assays, the authors showed that AGO2 is the major developmentally regulated Argonaute protein in mESCs, and that AGO2 is repressed by microRNA-182/microRNA-183. The experiments appear well-designed and the methods are technically sound. However, the study provides only marginal conceptual advances, especially given the very recent publication of a study by the same group showing how AGO2 is regulated by the heterochronic gene Trim71 (Liu et al., 2021, eLife). I have two major concerns for the authors to consider:

We appreciate that this reviewer believes that “the experiments appear well-designed and the methods are technically sound”. We provided our response to his/her specific comments below.

First, is there a way for the authors to confirm direct binding of microRNA-182/microRNA-183 to Ago2 RNA? If so, I consider this an essential experiment for the authors to carry out, given that the evidence thus far appears indirect (in sillico analysis showing presence of binding motifs and altered expression of Ago2 when the putative binding sites were mutated in cell lines).

We agree with the reviewer that showing miR-182/miR-183 associate with Ago2 mRNA will strengthen the conclusions of the manuscript.

To address this point, we performed a modified RNA anti-sense purification (RAP) approach (Figure 1—figure supplement 2A). Specifically, to trap potential transient and dynamic RNA-mediated interactions (e.g., microRNA-mediated regulation), we crosslinked the mESCs with 0.1% formaldehyde and then purified target mRNAs from the cell lysate using a set of biotinylated antisense DNA oligoes and streptatividin magnetic beads. The following observations from this experiment indicate that miR182/miR-183 specifically associate with Ago2 mRNA in mESCs.

First, the RAP approach can specifically isolate the target mRNAs (Figure 1—figure supplement 2B). Using anti-sense oligoes target Ago2 mRNAs, we observed Ago2 mRNAs, but not control mRNAs (e.g., Gapdh mRNA, Vim mRNAs), were significantly enriched in the RAP sample.

Second, we found that miR-182/miR-183 were specifically enriched in the RAP-Ago2 mRNA sample, but not RAP-Vim mRNA sample, arguing that these two miRNAs are associated with Ago2 mRNAs (Figure 1—figure supplement 2C).

Third, when the RAP was performed in the 3’UTR mutant mESCs, where the miR182/miR-183 binding sites were mutated (Figure 3A), we observed that the enrichment of miR-182/miR-183 in the RAP-Ago2 mRNA was significantly decreased (Figure 1—figure supplement 2C).

Altogether, these results indicate that miR-182/miR-183 specifically associated Ago2 mRNA in mESCs.

In the revised manuscript, we presented these results in Figure 1—figure supplement 2, and described the experimental procedures in the methods section.

Second, the authors have already shown in a previous work that Trim71 also represses Ago2. It is thus surprising to me that in the current study, the authors make no attempt to experimentally link how microRNA-182/microRNA-183 may be working together with Trim71 to regulate Ago2. What is the significance to the overall field to show identify and show additional repressors of Ago2, and how do these newly identified repressors cooperate with Trim71? It is essential for the authors to better link their current work in the context of previous findings by demonstrating how Ago2 repressors may be working together, and whether they may specific functions that distinguish them from each other.

We appreciate the reviewer’s great suggestion on improving our manuscript. We performed additional experiments to address his point.

Specifically, the different binding locations of Trim71 and miR-182/miR-183 in the 3’UTR of Ago2 mRNA led us to test whether or not the Trim71-mediated repression of Ago2 mRNA and the miR-182/miR-183-mediated repression of Ago2 mRNA functions independently in mESCs. We found that at the Ago2 expression level, in the absence of miR-182/miR-183-mediated repression (in the 3’UTR mutant mESCs), Trim71 still represses Ago2. Moreover, at the stem cell function level, we observed that inhibition of Trim71-mediated repression of Ago2 mRNA (through deleting the Trim71-binding site in the 3’UTR of Ago2 mRNA (CLIPD)) further decreased stem cell self-renewal and accelerated differentiation in the 3’UTR mutant mESCs. Collectively, these observations indicate that miR-182/miR-183 and Trim71 function in parallel to repress Ago2 mRNA in mESCs.

We presented these new results as Figure 5 and Figure 5 —figure supplement 1 and added a new section (from lane 176 to lane 197) in the revised manuscript.

From an experimental standpoint, the authors need to provide better mechanistic insights that explain how microRNA-182/microRNA-183 may cooperate with Trim71 to regulate Ago2 function in mESCs. Otherwise, the conceptual advance provided by the current manuscript appears minor.

This point is the same as the point 2 of the public review from this reviewer. Please see our response above.

Reviewer #2:

Liu et al. investigated the role of micorRNA(miRNA)-mediated regulation of miRNA-induced silencing complex (miRISC) during the differentiation of mouse embryonic stem cells (mESCs). Corroborating previous evidences establishing high mRNA levels for only one paralog of Argonautes (Ago2) in mESCs, the authors demonstrated that the Ago2 is expressed at high levels in mESCs at protein levels too, and that the AGO2 levels are elevated upon mESC differentiation. Using many independent and orthogonal experiments, the authors find with abundance of evidence that miRNAs, miR-182 and miR-183 whose conserved bindings are present in the 3'UTR of Ago2 and whose expression negatively correlates with AGO2 levels in mESCs directly repress AGO2 levels and that the loss of this miRNA-mediated repression of AGO2 promotes enhanced differentiation of mESCs in vitro. The strongest evidence of this direct regulation of AGO2 comes from the experiments where miR-182/183 sites are deleted in the AGO2 3'UTR (3'UTR mutant), which recapitulates the phenotype. While these results robustly show that AGO2/miR-182/183 axis enhances mESC differentiation during the induced transition of mESCs from the ground state to the primed state, this work doesn't show whether this axis is sufficient to control the differentiation in the ground/naive state. Finally, in concordance with their prior findings (Liu et al. 2021), the authors show that the AGO2 levels modulated by miR-182/183 also control the let-7-dependent differentiation of mESCs.

While the previous reports showed that miR-182/183 suppresses differentiation-inducing miRNAs, including let-7, miR-26 and miR-218, this work forms a unique niche and provides a link between miR-182/183 and let-7 in the form of miR-182/183-mediated repression of AGO2. The experiments in this paper follow a logical progression and provide high quality data. The conclusions are mostly supported by the data.

However, in its current form, this paper needs clarification on some fundamental aspects and some additional data. See below for the details.

We appreciate that this reviewer believes that “this work forms a unique niche” and “The experiments in this paper follow a logical progress and provide high quality data. The conclusions are mostly supported by the data”. We provided our response to his/her specific comments below.

1) The authors use as readout the reduction in colony formation during the transition of mESCs to the primed state, where the undifferentiated colonies are already reduced to 30%, and where there could be many confounding effects from large transcriptional changes. It is surprising that the authors didn't use the colony formation in naive/ground state as the readout and show that the increased AGO2 levels cause cells to lose stemness. They could potentially see much larger effects in the ground state, attributable directly to AGO2/miR-182/183. If using naive/ground state in these experiments is experimentally not feasible, the authors should discuss that.

The naïve/ground state mESCs were maintained through culturing mESCs in the presence of two chemical inhibitors: PD0325901 and CHIR-99021, which block mitogen-activated protein kinase (MEK1) and glycogen synthase kinase-3, respectively. These inhibitions potently block the stem cell differentiation, thereby maintaining mESCs in the naïve/ground state. Although this condition provides high percentage of AP+ colonies in the colony formation assay, the strong inhibition of differentiation by the chemical inhibitors will “mask” many pro-differentiation effects. Thus, the colony formation assay is usually performed in the 15% FBS + Lif medium, where both prostemness and pro-differentiation phenotypes can be observed.

In the revised manuscript, we add this rational so that general readers can understand why we did the stemness assays in the 15% FBS + Lif medium instead of the 2i+lif medium (line 109 of the revised manuscript).

2) Though the authors conclude that the elevated levels of AGO2 selectively stabilizes let-7 family members, the mechanism of this selectively is lacking. This model might be an over-simplification, since the increased AGO2 levels can cause system-wide cascade of direct and indirect effects, some of which might be responsible for this increased selectivity. It is likely that LIN28A, which is expressed at higher levels in mESCs and is involved in the well-established feedback loop with let-7 miRNA family, is involved in this let-7-specificity. A plausible alternative hypothesis is that the increased levels of AGO2 in 3'UTR mutant or during differentiation stabilizes all actively produced mature miRNAs equally. However, most miRNAs may not cause an immediate impact on levels of other miRNAs or the miRNA machinery, but the newly formed let-7-miRISCs reduce the high levels of LIN28A, promoting the elevated levels of mature let-7 miRNAs, which competes with other mature miRNAs and saturates the newly formed AGO2 molecules. This hypothesis doesn't assume that the increased AGO2 selectively stabilizes let-7 solitarily.

We appreciate this reviewer’s very insightful comments on the potential mechanisms by which the increased Ago2 specifically stabilizes and increases let-7 miRNAs in mESCs. Indeed, the data from our previous paper (Liu et al., 2021 eLife) indicated that Lin28a plays an important role in the let-7’s specific response to Ago2 levels, which is exactly as the reviewer predicted. Specifically, in Figure 5 of the previous eLife paper, we showed that when Ago2 level was induced, in addition to the specific increase of let7 mature miRNAs, there were also specific decrease of both Lin28a and Trim71, the two conserved (from C. elegans to human) targets of let-7 microRNAs. Thus, Ago2, let7 miRNAs, and Lin28a forms a delicate regulatory loop, which makes let-7 mature miRNA level sensitive to Ago2 levels. Specifically, a slight increase of the let-7 miRNAs caused by elevated Ago2 decreases Lin28a. This decrease alleviates Lin28a-mediated inhibition on the maturation of let-7 miRNAs, resulting in more let-7 pre-miRNAs become mature let-7 miRNAs, which further decreases Lin28a levels and prevents more let-7 pre-miRNA from Lin28a-mediated degradation. Thus, this positive regulatory loop amplifies let-7 miRNAs and makes the pro-differentiation let-7 miRNAs sensitive to Ago2 levels in stem cells. We made this point in the Discussion section of the previous eLife paper.

Although the regulatory loop involving Lin28a is the most likely explanation, we agree with the reviewer that it is difficult to identify the exact causal factor on why let-7 is specifically increased when Ago2 is elevated, using the in vivo system. Because as the reviewer nicely points out “the increased AGO2 levels can cause system-wide cascade of direct and indirect effects, some of which might be responsible for this increased selectivity.” We believe a complete resolution on this issue would require an in vitro system that can recapitulate these regulatory events, which can limit the potential system-wide cascade of indirect effects and enable specific and direct studies on the interplay among let-7 miRNA (pre-miRNA), Lin28a, Ago2, and Dicer (pre-miRNA processing machinery). Unfortunately, however, currently we don’t know such an in vitro system exists. Thus, we acknowledge the limitations in interpreting the results from the in vivo studies in mESCs.

As discussed above, our current work is closely related to the previous eLife paper. Thus, we feel the Research Advance format of eLife, which can be linked to the previous eLife paper, would be an ideal format for us to present these findings.

3) The authors claim that AGO2 repression via miR-182/183 is important for proper differentiation of mESC, however, the data in this paper doesn't show an impact of AGO2 repression on defective differentiation.

We feel the definition on “defective differentiation” mentioned by the reviewer is vague to us. Because “defective differentiation” can suggest many scenarios (e.g., mESCs do not differentiate at all, differentiate faster or slower, etc.). If we define the “defective differentiation” as any differentiation process that is different from the WT mESCs differentiation, then we believe that our results from this manuscript and the previous eLife paper shows that repressing Ago2 expression (by either miR-182/miR-183 in this study or Trim71 as described in the previous study) prevents mESCs from defective differentiation (as loss of these regulations accelerate the differentiation process of mESCs).

Some potential outstanding questions that the authors may want to explore towards this paper or the future work are:

We appreciate the reviewer’s thoughtful comments and suggestions on this work and on our future studies. We provide our response below.

1) How do miR-182/183 levels are modulated during the differentiation process and if modulating their levels is sufficient to promote differentiation in the ground/naive state;

First, previous genomic studies and CRISPR/Cas9-mediated functional studies (Pulecio et al., 2017; Rajagopal et al., 2016) indicate that at the transcriptional level, the production of miR-182 and miR-183 is regulated by the pluripotency factor Nanog in mESCs. These observations explain, in part (at the production level), why these miRNAs are highly expressed in mESCs.

Second, the ground state of mESCs is a transient developmental stage, and in culture, this state is normally maintained via the two chemical inhibitors (PD0325901 and CHIR99021). As discussed in the first point of the public review from this reviewer, these two chemical inhibitors “mask” many pro-differentiation effects. The following two observations (from our study and the literature), obtained from standard mESC culture conditions (15% FBS + lif), however, argues that inhibition of miR-182/miR-183 triggers differentiation: a) the miR-182/mi-183 knockout mESCs have very few AP+ colonies compared to the WT mESCs (Figure 2C); b) in Dgcr8 knockout mESCs, where normal differentiation is blocked due to the absence of endogenous miRNAs, miR-182/miR-183 can inhibit the strong differentiation effects induced by let-7 miRNAs (Wang et al., 2017). These results from both loss-of-function and gain-of-function studies strongly argue that decrease of miR-182/miR-183 levels can trigger differentiation in mESCs, and miR-182/miR-183 play important roles in maintaining pluripotency.

2) this paper suggests that the inhibition of many differentiation-inducing miRNAs that has been shown previously to be trigged by miR-182/183 (Wang et al., 2017), is orchestrated through miR-182/183 mediated suppression of miRISC machinery. It would be of great importance to investigate how do miR-182/183 themselves escape the effect of inhibited miRISC machinery, e.g., do these miRNAs have higher half-life in mESCs?

The reviewer raised a great question regarding regulations of miRISC in mESCs. The interpretation of the results could be: a) miR-182/183 have unique features that enable them to escape the effects of repressed miRISC machinery, as the reviewer suggested; or alternatively, b) among miRNAs expressed in mESCs, let-7s are uniquely sensitive to miRISC machinery.

To discriminate these two possibilities, we measured the stabilities of miR-183/miR-183 and let-7 miRNAs in mESCs maintained in the ground state (2i+lif) and differentiating mESCs (-lif medium) (Author response image 1):

Author response image 1

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These results indicate that there was no change of miR-182/miR-183 stability when mESCs were either in the ground state or in the differentiating state. Interestingly, however, let-7 miRNAs (let-7a, let-7f, miR-98) have significantly higher stability in differentiating mESCs than that in the ground state mESCs. This increase of stability correlates with Ago2 levels, as differentiating mESCs have higher Ago2 level than ground state mESCs have (Figure 1C). Combined with our previous observation that increase of Ago2 specifically increase and stabilize let-7 miRNAs in mESCs (Figure 5 and Figure 5 —figure supplement 2 in Liu et al., 2021 eLife), these observations argue that it is the let-7 miRNAs that are uniquely sensitive to Ago2 levels in mESCs.As the reviewer pointed out in the point 2) of the public review, and as we discussed in the previous eLife paper (Liu et al., 2021 eLife), this let-7 miRNAs’ unique sensitive to Ago2 levels could be due to the deliciated positive autoregulatory loops among let-7 miRNAs, Ago2, and Lin28a (a conserved let-7 target).

3) Does the loss of AGO2 repression only enhance the differentiation or does it also trigger defective differentiation?

As discussed in the point 3) of the public review, we feel that the meaning of “defective differentiation” is not clear to us. If “defective differentiation” is defined as any differentiation process that is different from the differentiation of WT mESCs, then loss of Ago2 repression results in accelerated mESC differentiation, which is a form of defective differentiation.

4) What is the significance of this regulation in vivo.

We completely agree with the reviewer that dissecting the in vivo relevance of this regulation (miR-182/miR-183 mediated regulation of Ago2) using animal models is of high significance for future work. Interestingly, miR-183 knockout mouse displays eye defects (Xiang et al., 2017), suggesting that absence of miR-182/miR-183 mediated regulation of Ago2 may contribute to these developmental defects.

One challenge of using animal models to determine the in vivo relevance of regulations of stemness is what phenotypes to look for in the genetically modified animals. The simplest scenario is the lethal phenotype, such as the Ago2 knockout mouse. Complicated scenarios include animals that do not have lethal phenotype but with some defects in certain tissues or cells, such as the Lin28a knockout mouse (Sato et al., 2020). We believe using cell-based experimental system to generate mechanistic insights at the molecular level will be of great help and significance to interpret the phenotypes of animal models.

"Two lines of evidence indicated that miR-182/miR-183 targets Ago2 mRNA." – should use "regulates" instead of "targets", since the evidence for direct targeting comes later in the paper.

We made this change as suggested by the reviewer.

References:

Pulecio, J., Verma, N., Mejia-Ramirez, E., Huangfu, D., and Raya, A. (2017). CRISPR/Cas9-Based Engineering of the Epigenome. Cell Stem Cell 21, 431-447.

Rajagopal, N., Srinivasan, S., Kooshesh, K., Guo, Y., Edwards, M.D., Banerjee, B., Syed, T., Emons, B.J., Gifford, D.K., and Sherwood, R.I. (2016). High-throughput mapping of regulatory DNA. Nat Biotechnol 34, 167-174.

Sato, T., Kataoka, K., Ito, Y., Yokoyama, S., Inui, M., Mori, M., Takahashi, S., Akita, K., Takada, S., Ueno-Kudoh, H., et al. (2020). Lin28a/let-7 pathway modulates the Hox code via Polycomb regulation during axial patterning in vertebrates. eLife 9.

Wang, X.W., Hao, J., Guo, W.T., Liao, L.Q., Huang, S.Y., Guo, X., Bao, X., Esteban, M.A., and Wang, Y. (2017). A DGCR8-Independent Stable MicroRNA Expression Strategy Reveals Important Functions of miR-290 and miR-183-182 Families in Mouse Embryonic Stem Cells. Stem Cell Reports 9, 1618-1629.

Xiang, L., Chen, X.J., Wu, K.C., Zhang, C.J., Zhou, G.H., Lv, J.N., Sun, L.F., Cheng, F.F., Cai, X.B., and Jin, Z.B. (2017). miR-183/96 plays a pivotal regulatory role in mouse photoreceptor maturation and maintenance. Proc Natl Acad Sci U S A 114, 6376-6381.

[Editors’ note: what follows is the authors’ response to the second round of review.]

Essential revisions:

Reviewer #3:

In this manuscript Liu and colleagues follow-up on their previous work showing that Ago2 expression is regulated in mESC by Trim-71 by proposing that Ago2 expression is also directly regulated by miR-182/miR-183 and that loss of this interaction affect mESC differentiation by resulting in let-7 stabilization.

Using elegant genetic and biochemical approaches they convincingly show that Ago2 is directly repressed by miR-183/miR-183, as either loss of these two miRNAs or mutation of their binding sites in the Ago2 3'UTR results in upregulation of Ago2 expression by approximately 2 fold.

They also show that targeted inactivation of miR-182/183 and, to a slightly lesser extent, mutation of miR-182/miR-183 binding sites in Ago2's 3'UTR promote differentiation of mESC in vitro.

The manuscript is very well written, the experiments are elegant and include all the appropriate controls, and their results are largely consistent with the model proposed by the authors. While the authors convincingly demonstrate that miR-182/182 directly regulate Ago2, it is slightly less clear whether the proposed increased activity of let-7 can fully explain the observed phenotype.

Overall this is a solid manuscript, although as discussed below not all conclusions are supported by conclusive evidence. I concur with reviewer 1 that the study provides relatively minor conceptual advance, but it can be argued that these results will be of interest to specialists in the field of miRNa and ESC biology.

A major concern I have regards the strength of the conclusion that the impaired stem cell function upon loss of miR-182/183 activity on Ago2 is mediated by increased activity of let-7. The LNA-based experiments are suggestive, but although they prove that blocking let-7 helps preventing differentiation, they do not prove that increased let-7 function underlies the observed phenotype in the Ago2 UTR mutant. In fact, the only evidence that let-7 activity is increased is a modest upregulation of let-7 members expression. Whether this is sufficient to affect gene expression to a detectable extent is unclear. The evidence would be strengthened substantially if the authors showed increased let-7-mediated gene repression in the UTR mutant. A straightforward and relatively inexpensive experiment would be to perform RNAseq analysis on the wt and UTR-mutant mESCs. The authors' model leads to the prediction that the a relatively selective de-repression of predicted let-7 targets should be observed in the UTR mutant samples.

We appreciated the reviewer’s insightful comment!

We did exactly the same experiment as the reviewer described in our previous paper on the mESCs with the Trim71 binding site deleted in the 3’UTR of Ago2 mRNA (Figure 4 in Liu et al., eLife, 2021). Through transcriptomic profiling via RNA-seq, we found that mRNAs with predicted let-7 binding sites showed increased level in the mutant mESCs than those in the WT mESCs compared to the mRNAs without let-7 binding sites, indicating increased let-7 miRNA activity (Figure 4E in Liu et al., eLife, 2021)

Here we used a different approach to address the same issue. Instead of examining mRNA levels, we measured protein level of an evolutionarily conserved let-7 target, Lin28a (the results are shown in Author response image 2). We observed that there was a decrease of Lin28a in the 3’UTR mutant mESCs. Moreover, there were no changes for Nanog and Oct4, which are not let-7 targets. There results argue that let-7 miRNA activity is increased in the 3’UTR mutant mESCs.

Author response image 2

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1) Figures 2C and 2D appear swapped in their description in the main text (lines 107-114)

We double checked Figures2C/2D and their corresponding descriptions in the main text, and we confirmed that they are in the correct order.

2) lines 132-135: I am not sure the authors can define the effect of the 3'UTR as not "in cis". Even if mediated by loss of miRNA binding, as is likely, the effect would still be on the transcript harboring the mutation, so technically, as far as I know, in cis. Perhaps a different language would be more appropriate. For example, the authors could say that the fact that deletion of miR-182/183 in a the 3'UTR-mut background doesn't result in additional upregulation of Ago2 provides additional evidence that the UTR mutation results in Ago2 upregulation by preventing miR-182/183 binding.

We made changes to correct the confusion caused by “in cis” in the revised manuscript (in line 132-134 of the revised manuscript).

3) It appears to me that loss of miR-182/183 has a stronger effect on ESC differentiation than mutation of their Ago2 3'UTR binding sites. This suggests that miR-182 promote ESC pluripotency by additional mechanism independent from Ago2. Perhaps the authors should comment on this in the discussion.

As the reviewer suggested, we added an additional paragraph (line 237-240) in the discussion commenting on this important implication.

https://doi.org/10.7554/eLife.72289.sa2

Article and author information

Author details

Qiuying Liu

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, United States

Contribution
Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0002-1474-4487
Mariah K Novak

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, United States

Contribution
Data curation, Investigation, Methodology, Writing - review and editing

Competing interests
No competing interests declared
Rachel M Pepin

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, United States

Contribution
Data curation, Investigation, Methodology, Writing - review and editing

Competing interests
No competing interests declared
Taylor Eich

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, United States

Contribution
Data curation, Investigation, Methodology, Writing - review and editing

Competing interests
No competing interests declared
Wenqian Hu

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, United States

Contribution
Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing - original draft, Writing - review and editing

For correspondence
hu.wenqian@mayo.edu

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0003-3577-3604

Funding

Mayo Foundation for Medical Education and Research

Qiuying Liu
Mariah K Novak
Rachel M Pepin
Taylor Eich
Wenqian Hu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Dr Xiaoli Chen for his assistance with miRNA prediction. This work is supported by Mayo Foundation for Medical Education and Research.

Senior Editor

James L Manley, Columbia University, United States

Reviewing Editor

Timothy W Nilsen, Case Western Reserve University, United States

Reviewer

Ravi K Patel, UCSF, United States

Version history

Preprint posted: July 21, 2021 (view preprint)
Received: July 21, 2021
Accepted: September 30, 2021
Accepted Manuscript published: October 1, 2021 (version 1)
Version of Record published: October 11, 2021 (version 2)

Copyright

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.