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. 2015 Jul 17;43(15):7577–7589. doi: 10.1093/nar/gkv728

Figure 2.

Figure 2.

ROCK inhibition enhances deadenylation of miRNA-targeted mRNAs. (A) Huh7 cells were treated with Y27632. Western blotting results shown are representative of two independent experiments. Similar results were obtained using Caco2 cells. (B) Levels of mature miR122 and pre-miR-122 were measured by northern blotting and normalized to U6 snRNA levels. Total RNA from lysates of non-transfected Huh7 cells or Huh cells transfected with a miR-122 precursor-expressing plasmid were used. nt, nucleotides. The results shown are representative of three independent experiments. (C) HepG2 cells were transfected with a miR-122 precursor-expressing plasmid. Y27632 was applied either simultaneously or 6 h before treatment with ActD. Levels of CAT-1 mRNA after ActD treatment were determined at the indicated time points by qRT-PCR. Data represent the means ± S.D. of three experiments. *P< 0.05. (D) The depression of miRNA target mRNA expression by Y27632 was miRNA-dependent. Levels of luciferase mRNA expressed from a luciferase reporter construct with miR-122-responsive elements in its 3′-UTR were determined after transfection with a miR-122 precursor-expressing plasmid into Dicer (–/–) (left panel) and Dicer (+/+) (right panel) MEFs as described in (C). Data represent the means ± S.D. of three experiments. *P< 0.05. (E) The effects of Y27632 were detected using natural target sites and endogenous miRNA. A luciferase reporter construct carrying a region of the 3′-UTR of Lin28B mRNA targeted by let-7 was transfected; luciferase mRNA levels were determined as described in (D). Data represent the means ± S.D. of three experiments. *P < 0.05. (F) Constructs used to determine the kinetics of deadenylation. β-globin ORFs with three wild-type let-7 target sites (let-7 wt) or mutated let-7 target sites (let-7 mut) were used. The transcription of these constructs in HeLa-Tet-off cells was regulated by doxycycline. (G, H) Northern blots showing deadenylation and decay of let-7 wt (G) and let-7 mut (H) β-globin constructs after transcription was stopped in cells that were pretreated (or not) with Y27632. Cells were treated with Y27632 for 6 h and then transcription from the constructs was stopped using doxycycline. β-actin mRNA levels were used for normalization. Representative blotting images are shown. Plots below the blotting images show the poly(A) shortening by indicating the distances from the position of poly(A)− and the β-globin reporter mRNA levels after normalization. Poly(A)− RNA was prepared in vitro by treating the RNA sample with oligo(dT) and RNase H. Data represent the means ± S.D. of five independent experiments. *P < 0.05.