Insulin-like growth factor 2 (IGF2), which regulates embryonic growth and plays a role in the progression of some tumors, is transcribed from multiple mRNA species that differ in their 5′ untranslated regions (5′UTRs). In cultured human rhabdomyosarcoma (RD) cells, the L4 form of IGF2 mRNA is constitutively translated in a rapamycin-insensitive manner, whereas translation of the L3 form can be inhibited by rapamycin. The IGF2 mRNA–binding proteins (IMPs) 1 to 3 modulate repression and stabilization of IGF2 mRNAs, and some have been implicated in tumor malignancy, yet a complete picture of their mechanism of action is lacking. Using reporter transcripts that mimicked the translational properties of L3 and L4, Dai et al. found that translation of the L3 transcript in RD cells occurs not through eIF-4E binding to the cap structure but rather through internal ribosome entry—and that rapamycin prevented translation of L3 by inhibiting binding of IMP2 to the transcript. IMP2 binding to L3, but not L4, was reduced in immunoprecipitates from rapamycin-treated cells, and in vitro binding studies indicated that rapamycin induced IMP2 dissociation from the 5′UTR of L3. Withdrawal of amino acids from the growth medium or treating RD cells with either rapamycin, which selectively inhibits the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), or the mTOR inhibitor Torin1 promoted binding of IMP2 to mTOR, suggesting that IMP2 was unable to bind to L3 in the absence of mTORC1 activity. IMP2 binding to mTOR was direct and, surprisingly, did not require raptor, which is involved in mTORC1 substrate recognition in other contexts. mTORC1 phosphorylated IMP2 in vitro, and IMP2 isolated from untreated cells was phosphorylated on Ser162, Ser164, or both. The doubly phosphorylated form was not detected in cells treated with rapamycin, and its abundance was greatly reduced by amino acid withdrawal. Experiments with mutant versions of IMP2 indicated that dual phosphorylation of these residues was required for IMP2 to promote translation of L3. The doubly phosphorylated form of IMP2 was present in mouse embryos, various adult mouse tissues, and human islets of Langerhans, suggesting that this mechanism of L3 translational stimulation by IMP2 might also operate in vivo. Genetic alterations in IMP2 and low birth weight are associated with an increased risk for type 2 diabetes in humans, and fetal nutrient deprivation coupled with postnatal nutrient excess increases the instance of diabetes in mice. Thus, these results reveal a mechanism by which nutrient availability may promote embryonic growth and contribute to type 2 diabetes.
N. Dai, J. Rapley, M. Angel, M. F. Yanik, M. D. Blower, J. Avruch, mTOR phosphorylates IMP2 to promote IGF2 mRNA translation by internal ribosomal entry. Genes & Dev. 25, 1159–1172 (2011). [Abstract] [Full Text]