Fig. 7.

miR-494 directly represses the NCL 3′UTR. (A) Schematic of plasmids pEGFP, pEGFP-NCL(3′), and pEGFP-NCL(3′mut), the latter bearing 6 mutant nucleotides in the NCL 3′UTR site corresponding to the miR-494 seed region. (B) HeLa cells were cotransfected with the plasmids shown in panel A and with either Ctrl siRNA or HuR siRNA, as indicated. Forty-eight hours after transfection, EGFP expression levels were assessed by Western blot analysis and quantified (left) and assessed by green fluorescence (right). The reporter EGFP protein expressed from pEGFP-NCL(3′) and pEGFP-NCL(3′mut) was slightly shorter because cloning of the 3′UTR regulatory sites required the removal of 16 amino acids from pEGFP and the introduction of a new stop codon. (C) The levels of EGFP mRNA in cells transfected as described in panel B were assessed by RT-qPCR analysis and normalized to GAPDH mRNA abundance. The data in panels B and C represent the means and standard deviations (s.d.) from 3 independent experiments. *, P < 0.05.