Figure 4. IMP1 binds to the 3′UTR of GDF15 mRNA and regulates the translation of its target mRNAs.

Aliquots of ³²P-labeled 3′ UTR of GDF15 mRNA were incubated with extracts prepared from MDA231/GFP and MDA231/GFP-IMP1 cells (A) and recombinant IMP1 (B). RNA-protein complexes (arrow indicated) were formed when the RNA probe incubated with IMP1-containing extracts and with recombinant IMP1. Cell extracts without IMP1 expressing did not form complex with the RNA probe. The complexes were competed by 500× excess of unlabeled 3′ UTR of GDF15 mRNA, but not the non-specific RNA (yeast tRNA). (C) Western blots showing the protein expression of four IMP1-bound mRNAs in MDA231/GFP and MDA231/GFP-IMP1 cells. Tubulin was used as a loading control. (D) Extracts of MDA231/GFP-IMP1 and MDA231/GFP cells were fractionated in 10–50% linear sucrose gradients. An OD₂₅₄ plot corresponding to the polysomal fractions was shown on the top panel. Total RNAs were isolated from the sucrose fractions. An equal amount of RNA from each fraction was subjected to Northern blots to detect the distribution of GDF15, PTGS2 and GAPDH mRNAs in the sucrose gradient.