Translational regulation by uORFs and start codon selection stringency

Abstract

In addition to the main, protein-coding, open reading frame (mORF), many eukaryotic mRNAs contain upstream ORFs (uORFs) initiated at AUG or near-cognate codons residing 5' of the mORF start site. Whereas translation of uORFs generally represses translation of the mORFs, a subset of uORFs serves as a nexus for regulating translation of the mORF. In this review, we summarize the mechanisms by which uORFs can repress or stimulate mRNA translation, highlight uORF-mediated translational repression involving ribosome queuing, and critically evaluate recently described alternatives to the delayed reinitiation model for uORF-mediated regulation of the GCN4/ATF4 mRNAs.

Keywords: ATF4; GCN4; eIF2 phosphorylation; ribosome queuing; stringency; uORF.

Figure 1.

uORF attributes that affect mORF translation include the following: (1) The efficiency of initiation at the uORF start codon, impacted by the start codon context sequence, controls leaky scanning. (2) The length of the uORF and the time it takes to translate the uORF affect the ability of a ribosome to resume scanning and reinitiate at a downstream start site following translation termination at the uORF stop codon. (3) The distance between the uORF stop codon and downstream start codon impacts the ability of a ribosome sliding down the mRNA after termination at the uORF stop codon to reacquire the initiation factors and Met-tRNA_i^Met required to initiate at a downstream start site.

Figure 2.

Schematic model of GCN4 translational control, simplified to show only uORF1 and uORF4, which are sufficient for nearly wild-type regulation. Following translation of uORF1 (boxed 1), posttermination 40S subunits remain attached to the GCN4 mRNA and resume scanning. (Left) Under nonstarvation conditions, they quickly rebind TC and reinitiate at uORF4 (boxed 4), and the 80S ribosome dissociates after terminating at uORF4. (Right) Under amino acid starvation conditions, the concentration of TC is reduced by eIF2α phosphorylation, such that many 40S ribosomes fail to rebind TC until after scanning past uORF4 and can thereby reinitiate at the GCN4 ORF instead. (Reproduced from Hinnebusch 2011 with permission from American Society for Microbiology).

Figure 3.

Schematic summary of the mRNA leader of GCN4 mRNA with its four short uORFs (uORF1 to uORF4), summarizing the positions and functions of elements surrounding uORF1 and uORF2 that promote reinitiation following their translation, including RPE(i) to RPE(iv) in the enhancer region upstream of uORF1 and the AU-rich motif following uORF1, as well as RPE(v) upstream of uORF2. RPE(i), RPE(iv), and RPE(v) interact with eIF3 (arrows) at the exit channel of the 40S subunit to enhance resumption of scanning by 40S posttermination complexes at the uORF stop codons. uORF3 and uORF4 allow much less reinitiation because they are closer to the GCN4 mORF, lack functional stimulatory elements found at uORF1 and uORF2, and contain the CCG Pro codon that impairs reinitiation. Additionally, the inefficient termination codon at uORF4 allows stop codon readthrough, placing posttermination 40S complexes even closer to the GCN4 mORF, thus rendering them less able to reinitiate there. (Reprinted with permission from Gunisova et al. 2016).

Figure 4.

uORF and stringency regulation of translation. (A) CHOP, GADD34, and AMD1 regulation. (Panels i,ii) Elongation/termination pause impairs leaky scanning over the uORF and represses CHOP and GADD34 translation, and eIF2α phosphorylation enhances leaky scanning to derepress mORF translation. (Panels iii,iv) Ribosomes access the vertebrate AMD1 mORF by leaky scanning over the cap-proximal uORF. Polyamine-triggered pausing of a ribosome on the uORF precludes additional ribosomes from loading, repressing mORF translation. (B) Alternative initiation enables bypass of a regulatory uORF. (Panel i) Initiation at upstream weak start sites in C/EBP mRNAs, generating LAP*, leads to ribosomes bypassing the short regulatory uORF that controls synthesis of LAP or LIP isoforms in response to eIF2α phosphorylation. (Panel ii) Reinitiation at the near-cognate start codon upstream of inhibitory uORF2 generates N-terminally extended cpc-1.

Figure 5.

Stringency control of eIF1 and eIF5 expression. Suboptimal AUG start codons of EIF1 mORF and inhibitory uORF in EIF5 mRNA sense global stringency of start codon selection: Low stringency enhances EIF1 mORF and EIF5 uORF translation, thereby increasing eIF1 and repressing eIF5 synthesis. High stringency increases leaky scanning over poor AUG codons, repressing EIF1 mORF and EIF5 uORF translation, thereby repressing eIF1 and increasing eIF5 synthesis.

Figure 6.

Schematic model of AZIN1 translational control. (Top) Under low-polyamine conditions, most ribosomes leaky-scan over the near-cognate AUU start codon of the uCC uORF and translate the mORF. (Bottom) Polyamine-triggered pausing of a ribosome on the PPW motif in the uCC causes ribosomes to queue and enhances initiation at the near-cognate uCC start codon, repressing AZIN1 synthesis.