Figure 4. Loss of Trim71-mediated repression of Ago2 mRNA translation results in significant post-transcriptional increase of let-7 miRNAs.

(A) Comparison of global miRNA expression in WT and CLIPΔ mouse embryonic stem cells (mESCs). The results are the average of four independent small RNA-seqs in the WT and the CLIPΔ mESCs. Blue dots: let-7 miRNAs; red dot: differentially expressed miRNAs; black dots: non-differentially expressed miRNAs. (B) qRT-PCR on let-7 miRNAs and non-let-7 miRNAs. For each miRNA, the expression level in WT cells was set as 1 for relative comparison. U6 RNA was used for normalization. (C) qRT-PCR on the let-7 pri-miRNAs and pre-miRNAs. For pri-miRNAs and pre-miRNAs, the expression level in the WT cells was set as 1 for relative comparison. 18S rRNA and U6 RNA were used for pri-miRNA and pre-miRNA normalization, respectively. The results in (B) and (C) are from three independent replicates. (D) Western blotting of Ago2, conserved let-7 targets, and non-let-7 targets. Gapdh was used for normalization in calculating the relative expression levels. (E) Cumulative distributions of expression level changes of let-7 targets, miRNA targets without let-7 binding sites, and mRNAs not targeted by miRNAs in WT and CLIPΔ mESCs.

Figure 4—figure supplement 1. The loss of Trim71-mediated repression of Ago2 mRNA translation does not alter global miRNA in mouse embryonic stem cells (mESCs).

(A) The relative miRNA expression pattern is highly similar between the WT and the CLIPΔ mESCs. Scatter plots show pairwise comparisons of the miRNA levels (log2 reads per million mapped reads, RPM) for annotated miRNAs from the four biological replicates. (B) Correlations of global miRNA expression patterns in the WT and the CLIPΔ mESCs determined by small RNA-seq from the four sets of biological replicates. (C) The relative levels of miRNAs. U6 RNA was used for normalization. The results represent the means (± SD) of three independent experiments. n.s. not significant (p>0.05) by the Student’s t-test.