Non-AUG translation: a new start for protein synthesis in eukaryotes

Abstract

Although it was long thought that eukaryotic translation almost always initiates at an AUG start codon, recent advancements in ribosome footprint mapping have revealed that non-AUG start codons are used at an astonishing frequency. These non-AUG initiation events are not simply errors but instead are used to generate or regulate proteins with key cellular functions; for example, during development or stress. Misregulation of non-AUG initiation events contributes to multiple human diseases, including cancer and neurodegeneration, and modulation of non-AUG usage may represent a novel therapeutic strategy. It is thus becoming increasingly clear that start codon selection is regulated by many trans-acting initiation factors as well as sequence/structural elements within messenger RNAs and that non-AUG translation has a profound impact on cellular states.

Keywords: RAN translation; eIF2A; eIF2D; near-cognate; start codon; translation initiation.

Figure 1.

Ribosome profiling can be used to reveal translation initiation sites across the transcriptome. (Left) Treatment with cycloheximide (blue), which binds to the E site of the 60S subunit, pauses all elongating 80S ribosomes. The mapped ribosome footprints thus typically cover the entire ORF. (Right) In contrast, harringtonine (red) binds to the A site of the 60S subunit and only inhibits 80S ribosomes just after subunit joining at the start codon. Mapped ribosome footprints from harringtonine treatment are thus enriched for the start codon.

Figure 2.

Canonical eukaryotic translation uses a scanning model of initiation. The 43S PIC consists of the 40S small ribosomal subunit, the TC (eIF2•GTP•Met-tRNA_i^Met), eIF1, eIF1A, eIF3, and eIF5. The PIC is recruited to the 5′ ends of mRNAs by the eIF4F complex, which consists of the cap-binding protein eIF4E, the DEAD-box helicase eIF4A, and the scaffolding protein eIF4G that binds PABP at the mRNA 3′ end. The PIC then scans in an ATP-dependent manner in a 5′-to-3′ direction in search of a start codon. Base pairing between the anti-codon of the Met-tRNA_i^Met and the start codon causes GTP hydrolysis by eIF2 and translation initiation. (1) eIF1; (1A) eIF1A; (4A) eIF4A; (4E) eIF4E; (4G) eIF4G.

Figure 3.

Canonical and alternative initiator tRNA_i-binding eIFs are differentially regulated and have different tRNA_i-binding stringency. (Left) Canonical translation uses eIF2 in a GTP-dependent manner to deliver the canonical Met-tRNA_i^Met to the P site of the 40S ribosomal subunit. This allows initiation at AUG start codons as well as at non-AUG start codons, albeit at a reduced efficiency. (Right) eIF2A and eIF2D are somewhat similar to eIF2 but are GTP-independent and have the capability to bind both charged and uncharged forms of tRNA_i^Met. eIF2A can also use Leu-tRNA^CUG and initiate at CUG and UUG start codons in some mRNAs in vitro and in vivo. eIF2D, which regulates reinitiation, can initiate at AUG codons in vitro as well as use other aa-tRNAs for initiation in a selective manner.

Figure 4.

Heat shock causes the production of a CUG-initiated MRPL18 protein that becomes incorporated into cytoplasmic ribosomes. (Top) Under normal growth conditions, the MRPL18 protein is synthesized from a canonical AUG start codon and includes the mitochondrial targeting signal (MTS; pink), which enables transport into mitochondria and subsequent incorporation into mitochondrial ribosomes. (Bottom) Heat shock causes a switch in the preferred start codon, resulting in initiation at a downstream CUG in the MRPL18 mRNA. The truncated MRPL18 (cyto) protein isoform lacks the MTS and is instead incorporated into cytoplasmic ribosomes, creating “hybrid ribosomes” that are required for increased Hsp70 mRNA translation during heat shock.

Figure 5.

eIF2A regulates translation of non-AUG uORFs in SCCs. (Top) In normal cells, translation of non-AUG uORFs is limited, and downstream ORFs are translated at basal levels. (Bottom) In some SCCs, cell stress and/or gene duplication events cause elevated eIF2A levels. This results in increased expression of non-AUG uORFs in oncogenic mRNAs (e.g., Ctnnb1) and subsequent up-regulation of the downstream oncoprotein ORFs. Upon depleting eIF2A in SCC models, premalignant and malignant progression is greatly reduced.

Figure 6.

RAN translation at CGG repeats in FMR1 produces neurotoxic proteins from multiple reading frames. The fragile X disorders are caused by a CGG nucleotide repeat expansion in the 5′ leader of the FMR1 gene, which encodes FMRP. Unaffected patients have a mean repeat length of ∼30 (top), whereas fragile X disorder patients have expanded repeats of >55 (bottom), and there is a correlation between repeat size and disease severity. CGG repeat expansion stimulates a noncanonical form of protein synthesis (called RAN translation) in multiple reading frames that uses a 5′ cap-, eIF4E-, and eIF4A-dependent scanning mode of initiation. RAN translation of the CGG repeat produces two toxic homopolymeric proteins from separate reading frames: (1) a polyglycine protein (FMRpolyG) that initiates at ACG and GUG codons upstream of the repeat in the +1 reading frame and (2) a polyalanine protein (FMRpolyA) that most likely initiates within the repeat at a GCG codon from the +2 reading frame.