An RNA stem-loop functions in conjunction with an upstream open reading frame to direct preferential translation in the integrated stress response

Abstract

In response to environmental stresses, cells invoke translational control to conserve resources and rapidly reprogram gene expression for optimal adaptation. A central mechanism for translational control involves phosphorylation of the α subunit of eIF2 (p-eIF2α), which reduces delivery of initiator tRNA to ribosomes. Because p-eIF2α is invoked by multiple protein kinases, each responding to distinct stresses, this pathway is named the integrated stress response (ISR). While p-eIF2α lowers bulk translation initiation, many stress-related mRNAs are preferentially translated. The process by which ribosomes delineate gene transcripts for preferential translation is known to involve upstream open reading frames (uORFs) embedded in the targeted mRNAs. In this study, we used polysome analyses and reporter assays to address the mechanisms directing preferential translation of human IBTKα in the ISR. The IBTKα mRNA encodes four uORFs, with only 5'-proximal uORF1 and uORF2 being translated. Of importance, the 5'-leader of IBTKα mRNA also contains a phylogenetically conserved stem-loop of moderate stability that is situated 11 nucleotides downstream of uORF2. The uORF2 is well translated and functions in combination with the stem-loop to effectively lower translation reinitiation at the IBTKα coding sequence. Upon stress-induced p-eIF2α, the uORF2/stem loop element can be bypassed to enhance IBTKα translation by a mechanism that may involve the modestly translated uORF1. Our study demonstrates that uORFs in conjunction with RNA secondary structures can be critical elements that serve as the "bar code" by which scanning ribosomes can delineate which mRNAs are preferentially translated in the ISR.