Figure 7.

The C-terminal deletion of Lin28 has a dominant-negative effect on the translation of endogenous Oct4 mRNA. Flag-Lin28ΔC or empty vector was transfected into PA-1 cells. Forty-eight hours later, protein and RNA were extracted and levels determined by western blot (A) and RT–qRCR (B), respectively. In (A), top blot, polyclonal antibodies against β-tubulin and Oct4 were simultaneously used to probe a single membrane piece that contained both Oct4 and β-tubulin. In the bottom blot, polyclonal anti-Lin28 antibody was used to detect a single piece of membrane containing both endogenous Lin28 and the transfected Flag-Lin28ΔC. Protein bands on western gels were quantitated using the Bio-Rad Quantity One software. (C) Distribution of Oct4 and β-actin mRNAs in the polysomes. Flag-Lin28ΔC or empty vector was transfected into PA-1 cells, and polysome fractionation carried out 48 h later. RNAs were extracted from each fraction and subjected to RT–qPCR using primers specific for Oct4, β-actin or β-tubulin (loading control) mRNA. The efficiency of translation was calculated as described in Figure 1 legend. Numbers are mean ± SD (n = 3), P < 0.01. (D) The C-terminus deletion of Lin28 does not affect its RNA-binding. Flag-Lin28, Flag-Lin28ΔC, or empty vector was transfected into PA-1 cells, IP RNP experiments using monoclonal anti-Flag M2 antibody were carried out 30 h after transfection. RNAs were extracted from isolated RNPs and subjected to RT–qPCR analysis. Levels of β-actin and Oct4 mRNAs in IP samples derived from vector-transfected cells were arbitrarily set as 1. Numbers are mean ± SD (n = 3). (E) Representative polysome profiles of PA-1 48 h after transfection of empty vector or Flag-Lin28ΔC.