Identification of AUF1 as a rapamycin-responsive binding protein to the 5′-terminal oligopyrimidine element of mRNAs

Abstract

In vertebrates, mRNAs containing a 5′-terminal oligopyrimidine (TOP) motif are coordinately post-transcriptionally regulated. Binding of specific proteins to this element has been proposed to downregulate expression of TOP mRNAs at the level of translational initiation. We previously reported that rapamycin induces binding activity to the TOP element of ribosomal protein (r-protein) L32 mRNA. In this study, we adapt DEAE-cellulose/oligo dT-cellulose tandem column chromatography to purify TOP element-binding proteins from bovine submaxillary lymph nodes (SLN). We also show by northwestern blot analysis that two proteins of molecular weight 47 kDa (47BP) and 43 kDa (43BP) specifically bind to a 32P-labeled riboprobe containing TOP regulatory element of the r-protein L32. Microsequencing of the purified 47BP revealed an internal sequence of 15 amino acids identical to the consensus sequence of the 2× RBD-Gly family. Western blot analysis of the cytoplasm fractions using an AUF1 antibody revealed that these two proteins are p45 AUF1 and p42 AUF1. Increases of the four isoforms of AUF1 protein were observed in 100,000 g supernatant fractions of rapamycin-administered rat SLN. Furthermore, decreases of p45 AUF1 and p42 and/or p40 AUF1 were observed in the polysomal fractions of BJAB cells in which translation of TOP mRNAs was selectively suppressed by rapamycin treatment. Taken together, these results suggest that AUF1 is a TOP mRNA-binding protein that may participate in the translational suppression of TOP mRNAs resulting from rapamycin treatment.