Figure 6: Trim71 cooperates with miR-302 to inhibit Cdkn1a expression.

(a) Trim71 depletion leads to upregulation of Cdkn1a. The same cell extracts from Fig. 1(d) were blotted with α-Cdkn1a antibody. (b) TaqMan PCR for mature miR-295 and miR-302a levels (normalized to snoRNA142). Error bars represent mean+/−s.d. with n=3. (c) J1 ESCs transfected with Trim71 siRNA or control siRNA, together with anti-miR-302a or control inhibitor, as indicated. 60 h post-transfection total RNA was analysed by qRT–PCR for Cdkn1a mRNA levels (normalized to β-actin). Error bars represent mean +/−s.d. with n=3. (d) Reporter assays were performed using a firefly luciferase that contains a miR-302 binding site from the mouse Cdkn1a 3′-UTR. A control pre-miRNA or pre-miR-302a was transfected into HeLa cells that do not express ESC-specific miRNAs. Signals were normalized to Renilla luciferase levels. Normalized signals for the pre-miR control were set to 100. (e) Reporter assays were performed 60 h post-transfection using the same luciferase constructs as in (d) as well as with a mutant reporter (white bars) in which nucleotides corresponding to the miR-302 seed sequence were mutated. Normalized signals for the mutant construct were set to 100 in each condition. Error bars represent mean +/−s.d. with n=3. The P-values are from two-tailed t-test.