Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells

Abstract

During cellular stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) elicits gene expression designed to ameliorate the underlying cellular disturbance. Central to this stress response is the transcriptional regulator activating transcription factor, ATF4. Here we describe the mechanism regulating ATF4 expression involving the differential contribution of two upstream ORFs (uORFs) in the 5 leader of the mouse ATF4 mRNA. The 5 proximal uORF1 is a positive-acting element that facilitates ribosome scanning and reinitiation at downstream coding regions in the ATF4 mRNA. When eIF2-GTP is abundant in nonstressed cells, ribosomes scanning downstream of uORF1 reinitiate at the next coding region, uORF2, an inhibitory element that blocks ATF4 expression. During stress conditions, phosphorylation of eIF2 and the accompanying reduction in the levels of eIF2-GTP increase the time required for the scanning ribosomes to become competent to reinitiate translation. This delayed reinitiation allows for ribosomes to scan through the inhibitory uORF2 and instead reinitiate at the ATF4-coding region. Increased expression of ATF4 would contribute to the expression of genes involved in remediation of cellular stress damage. These results suggest that the mechanism of translation reinitiation involving uORFs is conserved from yeast to mammals. During environmental stress conditions, cells induce a program of gene expression designed to remedy cellular damage or, alternatively, to elicit apoptosis. Central to the early events in stress response pathways is a family of protein kinases that phosphorylate the ␣ subunit of eukaryotic initiation factor-2 (eIF2) (1-10). In mammals, four eIF2 kinases have been identified, and each recognizes distinct stress signals and modulate downstream response pathways via translational control. These eIF2 kinases include general control nonderepressible-2 (GCN2) that is activated by nutritional stresses, dsRNA induced protein kinase (PKR), important for an antiviral defense pathway mediated by IFN, heme regulated inhibitor (HRI) that couples protein synthesis to the availability of heme in erythroid cells, and pancreatic eIF2 kinase, PEK (also known as Perk), important for remedying protein misfolding in the endoplasmic reticulum (ER). Phosphorylation of the ␣ subunit of eIF2 reduces the levels of eIF2-GTP available for translation initiation, contributing to lowered global protein synthesis concurrent with induced translational expression of genes that function to alleviate stress damage in cells.We have been interested in the molecular mechanisms by which selected mRNAs are translated in response to eIF2 phosphorylation. A classic example of such a stress remedy pathway involves the transcriptional activator GCN4 in Saccharomyces cerevisiae (1, 2). In yeast starving for nutrients, GCN2 phosphorylation of eIF2 induces translation of GCN4 mRNA. Translational expression of GCN4 occurs by a mechanism that involves four upstream ORFs (uORFs) in the 5Ј noncoding portion of the GCN4 mRNA. Hinneb...

Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

“…This mechanism is also used in the expression control of some important regulatory genes. (48)(49)(50)(51)(52)(53) For example, the mRNA of transcriptional factor SCL contains four start codons preceded by small uORF. It was demonstrated that ribosomes reach the first SCL start codon mostly by leaky scanning (skipping the uORF start codon), but the other three protein isoforms are synthesized by ribosomes reinitiating translation after termination at stop codon of uORF.…”

“…(54) This mechanism links the expression control of some important regulatory factors with cellular response to stress conditions through the eIF2 phosphorylation. (50,55) Thus, it seems likely that the low synthesis rate of some eukaryotic proteins is an important characteristic of their gene expression patterns. However, the regulatory role of uORFs in the selection of translation start sites is commonly not taken into account in gene annotation procedures.…”

p53 isoforms gain functions

“…p53 isoforms gain functions V Olivares-Illana and R Fåhraeus suppress cap-dependent translation, but will also allow cap-independent translation to take place in a selected number of mRNAs, such as ATF4 and cat-1, and IREScarrying mRNAs, such as p58 PITSLRE (Cornelis et al, 2000;Fernandez et al, 2002;Vattem and Wek, 2004). Interestingly, the latter shows similarities in mRNA translation control to p53 and p53/47 as the IRES of p58 PITSLRE is within the coding sequence of p110 PITSLRE .…”

“Translating” Tumor Hypoxia: Unfolded Protein Response (UPR)–Dependent and UPR-Independent Pathways

“…ATF4 is a transcription factor that initiates a crucial response during the UPR, being responsible for the activation of a large set of genes important for recovery and tolerance to ER and oxidative stress (39). Selective translation of ATF4 results from the presence of two conserved uORFs in its 5 ¶ UTR (100,101). uORFs act as a barrier to translation of the correct ORF because ribosome initiation at the start codon of the uORF prevents the ribosome from reaching the correct start codon.…”