SQSTM1/p62 transcript as a new target of the RNA-binding ELAVL1/HuR protein.

(A): RNA-binding activity of ELAVL1/HuR evaluated by AlphaScreen technology. Saturation binding experiments investigated by titrating a series of biotinylated single-stranded (BITEG-) RNAs, including TNFneg and SQSTM1/p62neg, that we designated as negative controls, against 1 nM of rELAVL1/HuR. Calculated dissociation constants (Kd) for TNFalpha (3.83±0.69 nM, R2 = 0.97), and SQSTM1/p62 (30.85±13.22 nM R2 = 0.91) are indicated. The plots represent mean ± SD of two independent experiments. (B): Saturation binding experiments as function of rELAVL1/HuR concentrations against four different type of RNA-substrates at 50 nM concentration. 1 nM of rELAVL1/HuR was enough to reach saturation of the binding. The plots represent Mean ± SD of two independent experiments. (C): Effect of MG-132 exposure on SQSTM1/p62 gene expression. Determination of SQSTM1/p62 mRNA by real-time qPCR in human ARPE-19 cells following treatments with solvent (control) or 5 µM MG-132 for 24 hrs. SQSTM1/p62 mRNA expression in control cells was taken as 100%. The values obtained from total cellular mRNA have been normalized to the level of RPL6 mRNA and expressed as mean ± S.E.M. ***pSQSTM1/p62 transcript increases in the cytoplasm following MG-132 stimulus. Fold enrichment detected by quantitative real-time RT-PCR of SQSTM1/p62 mRNA in control and 24 h MG-132 RPE cells following immunoprecipitation with anti-ELAV antibody (IP) in the cytoplasm. ***p