Fig 2: Trim71 represses Ago2 mRNA translation in mouse embryonic stem cells (mESCs).(A) Deletion of Trim71 binding region in Ago2 mRNA’s 3’UTR. Genotyping PCR was performed using the indicated P1 and P2 primers. CLIP?1 and CLIP?2 are two independent clones from the genomic editing. (B) Western blotting in the WT, CLIP?1, and CLIP?2 mESCs. (C) qRT-PCR quantification of two Trim71 target mRNAs, Ago2 mRNA, and Cdkn1a mRNA, in the WT, CLIP?1, and CLIP?2 mESCs. 18S rRNA was used for normalization. (D) Polysome analysis in WT and CLIP? mESCs. (E) Inhibiting Trim71’s binding on Ago2 mRNA specifically upregulates its translation. The mRNA distribution in the RNP, the 80S, and the polysome fractions (shown in C) were quantified by qRT-PCR in the WT and the CLIP? mESCs, respectively. (F) Overexpression of Trim71 and its mutants does not change Ago2 mRNA level in the WT mESCs. The expression level of Ago2 mRNA in the WT mESCs with an empty vector, FLAG-Trim71, FLAG-Trim71(C12A/C15A), and FLAG-Trim71(R738A) was quantified by qRT-PCR. 18S rRNA was used for normalization. (G) Quantification of the indicated mRNA distributions in the RNP, 80S, and polysome fractions in the cell lysates from the WT mESCs expressing an empty vector, FLAG-Trim71, FLAG-Trim71(C12A/C15A), or FLAG-Trim71(R738A). (H) Western blotting in WT mESCs expressing an empty vector, FLAG-Trim71, a Trim71 ubiquitination mutant (C12A/C15A), and a Trim71 RNA-binding mutant (R738A). (I) Western blotting in CLIP? mESCs expressing an empty vector, FLAG-Trim71, a Trim71 ubiquitination mutant (C12A/C15A), and a Trim71 RNA-binding mutant (R738A). The qPCR results in (C) and (E–G) represent the means (± SD) of three independent experiments. *p<0.05, and n.s. not significant (p>0.05) by the Student’s t-test.

Fig 3: Inhibiting let-7 miRNAs blocks the stemness defects caused by the loss of Trim71-mediated repression of Ago2 mRNA translation.(A) Western blotting in WT and CLIP? mouse embryonic stem cells (mESCs) expressing GFP, V5-Lin28a, or Lin28b. Beta-tubulin was used for normalization in determining the relative expression level of let-7 targets Lin28a and Trim71. (B) Relative levels of miRNAs. U6 RNA was used for normalization. The results represent the means (± SD) of four biological replicates. (C) Colony formation assay for WT and CLIP? mESCs expressing GFP, V5-Lin28a, or Lin28b. (D) Exit pluripotency assay for WT and CLIP? mESCs expressing GFP, V5-Lin28a, or Lin28b. The results in (C) and (D) represent the means (± SD) of six independent experiments. (E) Western blotting of pluripotency factors during EB formation at Day 0 and Day 5 of WT and CLIP? mESCs expressing GFP, V5-Lin28a, or Lin28b. (F) Colony formation assay for WT and CLIP? mESCs cultured in the presence of 500 nM anti-let-7 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.

Fig 4: Trim71 represses mRNA translation at post-initiation step(s) in mouse embryonic stem cells (mESCs).(A) FLuc reporters for the tethering assay. (B) The FLuc activity and mRNA level determined in the tethering assay. (C) The IRES-containing bicistronic reporters. (D) mRNA levels from the IRES-containing reporters. (E) Luciferase activities from the IRES-containing reporters. (F) The poly(A) minus FLuc reporters. (G) mRNA levels from the poly(A) minus reporters. (H) FLuc activities from the poly(A) minus reporters. The results represent the means (± SD) of three independent experiments. *p<0.05, and ns. not significant (p>0.05) by the Student’s t-test.

Fig 5: Trim71-mediated repression of Ago2 mRNA translation is required for maintaining pluripotency.(A) Colony formation assay for mouse embryonic stem cells (mESCs). The mESCs were cultured in 15%FBS + Lif for 7 days, and the resultant colonies were fixed and stained for AP. (B) 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 (A) and (B), 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 four independent experiments. *p<0.05, and n.s. not significant (p>0.05) by the Student’s t-test. (C) Western blotting of pluripotency factors during EB formation. (D) Western blotting of markers of lineage-committed cells during mESC monolayer differentiation.