The mRNA Elements Directing Preferential Translation in the Integrated Stress Response

Abstract

In response to environmental and physiological stresses, cells impose translational control to reprogram adaptive gene expression and conserve energy and nutrients. A central mechanism regulating translation involves phosphorylation of the a-subunit of the eukaryotic initiation factor -2 (p-eIF2a), which reduces delivery of initiator tRNA to ribosomes and represses global protein synthesis. The pathway featuring p-eIF2a is called the integrated stress response because it involves multiple related eIF2a kinases, each responding to different stress arrangements. While p-eIF2a limits global protein synthesis, a subset of mRNAs are preferentially translated in response to p-eIF2a. Preferential translation of stress adaptive mRNAs is regulated by upstream opening reading frames (uORFs) present in the 5’-leader of these transcripts. In most cases uORFs are inhibitory in nature, but in some case uORFs can instead promote the translation of the downstream CDS. This study is focused on preferential translation of the gene Inhibitor of Bruton’s Tyrosine Kinase-alpha (IBTKa) in response to endoplasmic reticulum stress. The human IBTKa gene encodes a 1353 amino acid residue protein, along with a 5’-leader featuring predicted canonical uORFs. Among the four predicted uORFs, the 5'-proximal uORF1 and uORF2 are phylogenetically conserved among mammals and are well translated as judged by reporter assays, whereas uORF3 and uORF4 are not conserved and are poorly translated. In addition to the uORFs in the IBTKa mRNA, a phylogenetically conserved stem-loop (SL) of moderate stability is present 11 nucleotides downstream of uORF2. Using luciferase reporter assay, the uORF2 and SL were shown to function together to repress the translation of human IBTKa. In non-stressed conditions, the SL combined with uORF2 are critical for reducing ribosomes from reinitiating at the IBTKa coding sequence (CDS), thus repressing IBTKa expression. Upon ER stress and induced p-eIF2a, the more modestly translated uORF1 facilitates the bypass of the inhibitory uORF2/SL to enhance the translation of main CDS of IBTKa. This study demonstrates that uORFs in conjunction with RNA secondary structures can be critical elements that serve as a “bar code” by which scanning ribosomes decide which mRNAs are preferentially translated in the integrated stress response.