The eukaryotic 5' untranslated region (UTR) is critical for ribosome recruitment to the messenger RNA (mRNA) and start codon choice and plays a major role in the control of translation efficiency and shaping the cellular proteome. The ribosomal initiation complex is assembled on the mRNA via a cap-dependent or cap-independent mechanism. We describe various mechanisms controlling ribosome scanning and initiation codon selection by 5' upstream open reading frames, translation initiation factors, and primary and secondary structures of the 5'UTR, including particular sequence motifs. We also discuss translational control via phosphorylation of eukaryotic initiation factor 2, which is implicated in learning and memory, neurodegenerative diseases, and cancer.

Stop codon readthrough is used extensively by viruses to expand their gene expression. Until recent discoveries in Drosophila, only a very limited number of readthrough cases in chromosomal genes had been reported. Analysis of conserved protein coding signatures that extend beyond annotated stop codons identified potential stop codon readthrough of four mammalian genes. Here we use a modified targeted bioinformatic approach to identify a further three mammalian readthrough candidates. All seven genes were tested experimentally using reporter constructs transfected into HEK-293T cells. Four displayed efficient stop codon readthrough, and these have UGA immediately followed by CUAG. Comparative genomic analysis revealed that in the four readthrough candidates containing UGA-CUAG, this motif is conserved not only in mammals but throughout vertebrates with the first six of the seven nucleotides being universally conserved. The importance of the CUAG motif was confirmed using a systematic mutagenesis approach. One gene, OPRL1, encoding an opiate receptor, displayed extremely efficient levels of readthrough (∼31%) in HEK-293T cells. Signals both 5′ and 3′ of the OPRL1 stop codon contribute to this high level of readthrough. The sequence UGA-CUA alone can support 1.5% readthrough, underlying its importance.

In eukaryotes, it is generally assumed that translation initiation occurs at the AUG codon closest to the messenger RNA 5′ cap. However, in certain cases, initiation can occur at codons differing from AUG by a single nucleotide, especially the codons CUG, UUG, GUG, ACG, AUA and AUU. While non-AUG initiation has been experimentally verified for a handful of human genes, the full extent to which this phenomenon is utilized—both for increased coding capacity and potentially also for novel regulatory mechanisms—remains unclear. To address this issue, and hence to improve the quality of existing coding sequence annotations, we developed a methodology based on phylogenetic analysis of predicted 5′ untranslated regions from orthologous genes. We use evolutionary signatures of protein-coding sequences as an indicator of translation initiation upstream of annotated coding sequences. Our search identified novel conserved potential non-AUG-initiated N-terminal extensions in 42 human genes including VANGL2, FGFR1, KCNN4, TRPV6, HDGF, CITED2, EIF4G3 and NTF3, and also affirmed the conservation of known non-AUG-initiated extensions in 17 other genes. In several instances, we have been able to obtain independent experimental evidence of the expression of non-AUG-initiated products from the previously published literature and ribosome profiling data.

The central feature of standard eukaryotic translation initiation is small ribosome subunit loading at the 5′ cap followed by its 5′ to 3′ scanning for a start codon. The preferred start is an AUG codon in an optimal context. Elaborate cellular machinery exists to ensure the fidelity of start codon selection. Eukaryotic initiation factor 1 (eIF1) plays a central role in this process. Here we show that the translation of eIF1 homologs in eukaryotes from diverse taxa involves initiation from an AUG codon in a poor context. Using human eIF1 as a model, we show that this poor context is necessary for an autoregulatory negative feedback loop in which a high level of eIF1 inhibits its own translation, establishing that variability in the stringency of start codon selection is used for gene regulation in eukaryotes. We show that the stringency of start codon selection (preferential utilization of optimal start sites) is increased to a surprising degree by overexpressing eIF1. The capacity for the cellular level of eIF1 to impact initiation through the variable stringency of initiation codon selection likely has significant consequences for the proteome in eukaryotes.Kozak context | non-AUG | IF3

An AUG in an optimal nucleotide context is the preferred translation initiation site in eukaryotic cells. Interactions among translation initiation factors, including eIF1 and eIF5, govern start codon selection. Experiments described here showed that high intracellular eIF5 levels reduced the stringency of start codon selection in human cells. In contrast, high intracellular eIF1 levels increased stringency. High levels of eIF5 induced translation of inhibitory upstream open reading frames (uORFs) in eIF5 mRNA that initiate with AUG codons in conserved poor contexts. This resulted in reduced translation from the downstream eIF5 start codon, indicating that eIF5 autoregulates its own synthesis. As with eIF1, which is also autoregulated through translation initiation, features contributing to eIF5 autoregulation show deep evolutionary conservation. The results obtained provide the basis for a model in which auto- and cross-regulation of eIF5 and eIF1 translation establish a regulatory feedback loop that would stabilize the stringency of start codon selection.

Translation initiation is typically restricted to AUG codons, and scanning eukaryotic ribosomes inefficiently recognize near-cognate codons. We show that queuing of scanning ribosomes behind a paused elongating ribosome promotes initiation at upstream weak start sites. Ribosomal profiling reveals polyamine-dependent pausing of elongating ribosomes on a conserved Pro-Pro-Trp (PPW) motif in an inhibitory non-AUG-initiated upstream conserved coding region (uCC) of the antizyme inhibitor 1 (AZIN1) mRNA, encoding a regulator of cellular polyamine synthesis. Mutation of the PPW motif impairs initiation at the uCC's upstream near-cognate AUU start site and derepresses AZIN1 synthesis, whereas substitution of alternate elongation pause sequences restores uCC translation. Impairing ribosome loading reduces uCC translation and paradoxically derepresses AZIN1 synthesis. Finally, we identify the translation factor eIF5A as a sensor and effector for polyamine control of uCC translation. We propose that stalling of elongating ribosomes triggers queuing of scanning ribosomes and promotes initiation by positioning a ribosome near the start codon.

Previous studies with mice overproducing ornithine decarboxylase have demonstrated the importance of polyamine homeostasis for normal mammalian spermatogenesis. The present study introduces a likely key player in the maintenance of proper polyamine homeostasis during spermatogenesis. Antizyme 3 is a paralog of mammalian ornithine decarboxylase antizymes. Like its previously described counterparts, antizymes 1 and 2, it inhibits ornithine decarboxylase, which catalyzes the synthesis of putrescine. Earlier work has shown that the coding sequences for antizymes 1 and 2 are in two different, partially overlapping reading frames. Ribosomes translate the first reading frame, and just before the stop codon for that frame, they shift to the second reading frame to synthesize a trans-frame product. The efficiency of this frameshifting depends on polyamine concentration, creating an autoregulatory circuit. Antizyme 3 cDNA has the same arrangement of reading frames and a potential shift site with definite, although limited, homology to its evolutionarily distant antizyme 1 and 2 counterparts. In contrast to antizymes 1 and 2, which are widely expressed throughout the body, antizyme 3 transcription is restricted to testis germ cells. Expression starts early in spermiogenesis and finishes in the late spermatid phase. The potential significance of antizyme 3 expression during spermatogenesis is discussed in this paper.

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