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Published in final edited form as: Exp Cell Res. 2020 Mar 21;391(1):111973. doi: 10.1016/j.yexcr.2020.111973

Non-AUG start codons: expanding and regulating the small and alternative ORFeome

Xiongwen Cao †,‡, Sarah A Slavoff †,‡,⊥,*
PMCID: PMC7256928  NIHMSID: NIHMS1579949  PMID: 32209305

Abstract

Recent ribosome profiling and proteomic studies have revealed the presence of thousands of novel coding sequences, referred to as small open reading frames (sORFs), in prokaryotic and eukaryotic genomes. These genes have defied discovery via traditional genomic tools not only because they tend to be shorter than standard gene annotation length cutoffs, but also because they are, as a class, enriched in sequence properties previously assumed to be unusual, including non-AUG start codons. In this review, we summarize what is currently known about the incidence, efficiency, and mechanism of non-AUG start codon usage in prokaryotes and eukaryotes, and provide examples of regulatory and functional sORFs that initiate at non-AUG codons. While only a handful of non-AUG-initiated novel genes have been characterized in detail to date, their participation in important biological processes suggests that an improved understanding of this class of genes is needed.

Keywords: Genomics, proteomics, Non-AUG start codon, small ORF, sORF

Introduction

Over the past decade, advances in genomic and proteomic technologies1 have accelerated the discovery of thousands of small open reading frames of fewer than 100 codons (sORFs)2–7 in genomes spanning evolutionary space. These ORFs were previously unannotated due to size cutoffs and other assumptions applied by genome annotation consortia8,9, and are encoded in RNA regions previously assumed to be non-coding (e.g., long non-coding RNAs, 5′ and 3′ untranslated regions (UTRs)) as well as “alt-ORFs” overlapping annotated protein coding sequences or main ORFs3,6. We will collectively refer to recently discovered, short genes as sORFs. These newly discovered ORFs can be translated into polypeptide products that we will refer to as microproteins, though nomenclature has varied in prior reports10–12. The vast majority of recently reported sORFs remain uncharacterized, and some may represent protogenes13 or cis-translational regulators rather than encoding functional microproteins. However, an increasing number of sORFs have been shown to play important roles in biological processes, including regulation of protein complexes, membrane channels, and transcription factors, among others14.

Surprisingly, analyses of sORF coding sequences have revealed that these genes escaped annotation not only because they are short, but because, in many cases, they circumvent additional assumptions about the structure of genes. For example, sORFs overlapping main ORFs in alternative reading frames have been reported in both prokaryotic and eukaryotic genomes; examples include human DEDD23 and E. coli gndP19. Even more strikingly, several reports estimate that up to approximately 50% of sORFs detected to date do not initiate with canonical AUG start codons2,3,7,16,20. These findings suggest that information may be more densely encoded in non-viral genomes than previously assumed, and that assumptions about the size, sequence and monocistronic nature of eukaryotic genes provide an incomplete picture. In this minireview, we discuss the ubiquity and functions of sORFs that initiate with non-AUG start codons.

Genome-wide frequency of initiation at non-AUG start codons

Translation initiation of main ORFs at non-AUG start codons has recently been extensively reviewed21,22, so we provide only a brief overview here. Eukaryotic genes were previously assumed to initiate specifically at the first AUG start codon of a transcript, in accordance with the scanning model of translation initiation22. Occasional reports of initiation of previously known eukaryotic proteins at upstream or downstream, in-frame non-AUG start codons, such as FGF-2, which initiates at a CUG codon23,24, and c-myc, which can exist as an N-terminally extended isoform via initiation at an upstream in-frame CUG25,26, did little to change the perception that these events were rare. In contrast, prokaryotic genes have long been known to initiate at non-AUG start codons; for example, an analysis of 620 bacterial genomes revealed that ~80% of annotated genes initiate at AUG codons, ~12% at GUG and ~8% at UUG, with variable incidences of AUU and AUC across species27. The advent of ribosome profiling, or deep sequencing of ribosome-protected RNA footprints as a readout of translation, revealed a much more complex picture of eukaryotic translation initiation: hundreds to thousands of short, unannotated upstream open reading frames in the 5′ leaders of yeast transcripts were found to initiate with near-cognate start codons2, particularly during meiosis28. Application of inhibitors of translation initiation in bacteria, retapamulin and Onc112, have recently been similarly applied to identification of translation initiation sites and dozens of sORFs in bacteria29, revealing abundant initiation at GUG, UUG, CUG and AUU codons, as well as occasional utilization of AUC15 (Table 1A). Analogous inhibitors of translation initiation in mammalian cells, such as harringtonine20 and lactidomycin16,30, have similarly revealed that approximately 50% of all translation initiation events (including both canonical ORFs and sORFs) occur at non-AUG start codons (Table 1B), with CUG appearing as the most common near-cognate initiation codon (15–16% of initiation sites16).

Table 1. Frequency and efficiency of non-AUG start codon utilization in bacterial and eukaryotic cells from selected sources.

Observed frequency of non-AUG start codon occupancy by ribosome profiling in (A) retapamulin-treated bacterial cells15 and (B) lactidomycin-treated human and mouse cells16. Efficiency of non-AUG start codon use, expressed as percentage AUG efficiency, in (C) a GFP reporter gene assay inside living bacterial cells17 and (D) a firefly luciferase reporter gene assay in Neurospora crassa18.

A U C A G B U C A G
U 0.04% NR 0.04% NR U U 0.22% 0.08% NR NR U
0.09% NR 0.02% NR C 0.29% 0.13% 0.23% NR C
0.06% NR 0.02% NR A 0.09% 0.09% 0.01% NR A
5.62% NR NR NR G 4.17% 0.13% <0.01% NR G
C NR NR NR NR U C 0.05% 0.11% NR 0.07% U
NR NR NR 0.02% C 0.25% 0.16% 0.25% 0.42% C
0.02% NR 0.06% NR A 0.16% 0.28% 0.07% 0.12% A
3.12% 0.02% NR NR G 15.44% 0.29% 0.51% 0.11% G
A 6.18% NR 0.02% 0.02% U A 3.05% 0.13% NR 0.04% U
4.12% 0.06% 0.04% 0.04% C 2.71% 0.42% 0.32% 0.18% C
0.02% 0.06% 0.13% 0.06% A 1.29% 0.17% 1.34% 0.15% A
69.47% 0.02% 0.02% 0.02% G 49.76% 3.89% 1.00% 0.91% G
G NR 0.02% NR 0.02% U G NR 0.22% NR 0.10% U
NR NR NR NR C 0.25% 0.49% 0.31% 0.39% C
0.07% 0.02% 0.02% NR A 0.12% 0.32% 0.31% 0.31% A
10.40% 0.06% 0.02% 0.02% G 7.17% 0.38% 0.44% 0.10% G
C U C A G D U C A G
U 0.10% 0.11% 0.58% 0.07% U U NR NR NR NR U
0.05% 0.07% 0.10% 0.07% C NR NR NR NR C
0.18% 0.08% 0.09% 0.08% A NR NR NR NR A
63.99% 0.11% 0.10% 0.17% G 8.30% NR NR NR G
C 0.04% 0.05% 0.39% 0.07% U C NR NR NR NR U
0.08% 0.10% 0.11% 0.08% C NR NR NR NR C
0.11% 0.06% 0.06% 0.06% A NR NR NR NR A
2.72% 0.05% 0.05% 0.06% G 18.20% NR NR NR G
A 1.92% 0.12% 0.35% 0.08% U A 5.90% NR NR NR U
1.03% 0.11% 0.08% 0.07% C 2.90% NR NR NR C
1.84% 0.10% 0.10% 0.09% A 5.20% NR NR 0.01% A
100.00% 0.42% 0.05% 0.10% G 100.00% 4.50% 0.01% 0.02% G
G 0.55% 0.13% 0.34% 0.17% U G NR NR NR NR U
0.15% 0.07% 0.15% 0.11% C NR NR NR NR C
0.63% 0.15% 0.43% 0.37% A NR NR NR NR A
121.84% 0.26% 0.23% 0.23% G 11.40% NR NR NR G
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Mass spectrometry studies have hinted that sORFs initiate with non-AUG start codons more often than main ORFs in eukaryotic cells. Multiple studies, to date, have aimed to revise the initiation sites of main ORFs, revealing N-terminal extensions initiating from upstream, in-frame non-AUG start codons; however, the numbers of these N-terminal extensions identified in a given study is typically <100 (e.g., 42 by Baranov et al. using a combined computational and experimental approach; 50 by Zhu et al. using a mass spectrometry-based proteogenomics approach; 17 by Van Danne et al. using an N-terminalomics approach combined with ribosome profiling), a small fraction of the ~20,000 annotated human proteins. In contrast, mass spectrometry studies specifically designed for sORF enrichment and detection have revealed that up to ~50% of sORFs initiate at non-AUG start codons3,6,31,32, though it is important to note that start codons must be indirectly inferred from bottom-up proteomics data. More quantitatively, ribosome profiling has provided direct comparisons of the non-AUG start codon usage between annotated proteins and sORFs in mammalian cells. For example, Ingolia and colleagues20 identified 5647 start codons for canonical mouse proteins, including N-terminal truncations and extensions; as a class, these canonical proteins were relatively large, exceeding 100 amino acids 93% of the time. Of these, 79% utilized AUG and 21% utilized near-cognate start codons. In the same study, sORFs (which include ORFs mapping to “non-coding” RNAs; ORFs encoded in 5′ UTRs; ORFs internal and out-of-frame with annotated protein coding sequences; and polycistronic/downstream ORFs) were generally (96%) shorter than 100 amino acids, and utilized non-AUG start codons 70% of the time. Similarly, Qian and colleagues reported that global translation initiation sites, including both sORFs and canonical ORFs, occur at non-AUG triplets 50% of the time, while uORFs (upstream ORFs, or sORFs that occur in 5′ UTRs) initiate at near-cognate start codons with a remarkable 74% frequency16. While the absolute frequencies of AUG vs. non-AUG initiation vary in each study, the trend toward increased incidence of near-cognate start codons in the sORFeome relative to main ORFs is consistently observed.

Efficiency and mechanism of initiation at non-AUG start codons

Long before the genome-wide ubiquity of non-AUG initiation sites was recognized, the ability of both prokaryotic and eukaryotic ribosomes to initiate at near-cognate start codons was demonstrated, though at fractional efficiencies of initiation at AUG (Table 1C–D). In prokaryotes, a classic study demonstrated that “class I” UUG and GUG codons can initiate translation in E. coli with 12–15% the efficiency of an AUG within the same reporter construct; “class IIA” start codons CUG, AUU, AUC, AUA and ACG produced 1–3% protein yields relative to AUG, and the remaining codons tested (AGG, AAG) produced no detectable translation33. A more recent study using high-sensitivity reporters remarkably detected some level of translation initiation from almost all 64 codons (Table 1C), though sequences supporting the highest initiation efficiencies followed similar trends to previous reports, and a strong preference for U at the second position was observed17. An important early study in eukaryotic cell-free translation systems34 revealed translation initiation from CUG, GUG, ACG, AUA, UUG, AUU and AUC codons with 36–82% the efficiency of AUG in rabbit reticulocyte lysates and 8–45% in wheat germ lysate, though the efficiency of initiation at these triplets is likely to be different in living cells. More recent studies using a high-sensitivity reporter revealed translation from these codons with 2.9–18.2% the efficiency of AUG in rabbit reticulocyte lysates18, and 1.7–19.5% in 293T cells35. These reporter assay-based results are consistent with the genome-wide studies, suggesting that non-AUG initiated translation is frequently and efficiently utilized from bacteria to human.

The mechanism of initiation at non-AUG start codons differs in prokaryotes and eukaryotes, as does translation initiation at standard AUG start codons. In bacteria, assembly of the 30S preinitiation complex requires a Shine-Dalgarno sequence upstream of an AUG or near-cognate start codon. This preinitiation complex positions the start codon in the P-site, base-paired with the anticodon of the initiator fMet-tRNA (N-formylmethionine-tRNA); subsequent steps permit formation of the 70S initiation complex and protein synthesis and have been reviewed elsewhere36. A crystal structure of a T. thermophilus 70S initiation complex bound to fMet-tRNA base-paired to a near-cognate AUC codon in the P-site reveals that accommodation of the near-cognate-initiator tRNA codon-anticodon mismatch occurs through a wobble mechanism37. The relative efficiencies of AUG vs. near-cognate start codon utilization are primarily controlled by bacterial initiation factor 3 (IF3)33.

In eukaryotes, most translation initiation occurs via a scanning mechanism that begins when the 43S preinitiation complex consisting of the small ribosomal subunit, multiple initiation factors, PABP, and the ternary complex of eIF2 bound to GTP and the initiator tRNA recognize the mRNA 7-methylguanosine cap, then scan 5′-to-3′ until the first start codon is recognized22. eIF2 then hydrolyzes GTP and translation initiation occurs. Classic radiolabeling studies demonstrated that methionine is incorporated via scanning initiation at non-AUG start codons34, suggesting that wobble base pairing between the initiator tRNA and near-cognate codons occurs. The presence of a Kozak consensus sequence (especially an A or G at the −3 position) strongly affects the efficiency of initiation at non-AUG start codons in yeast and human cells3,38. More efficient initiation at AUG relative to near-cognate start codons is maintained in eukaryotic cells by translation initiation factors. In particular, mutations in eIF1 that decrease its affinity for eIF2β increase the relative efficiency of binding of the ternary complex to a near-cognate UUG start codon39. There are some exceptions to the scanning initiation mechanism at non-AUG start codons; for example, in select cases, initiation can occur at CUG codons using leucyl-tRNA and eIF2A in place of eIF240. A more recently reported class of non-AUG translation events, collectively referred to as RAN (repeat-associated non-AUG) translation, is responsible for translation of toxic dipeptide repeat proteins from microsatellite expansions associated with various diseases including Huntington’s disease41, fragile X-associated tremor ataxia syndrome42, and amyotrophic lateral sclerosis/frontotemporal dementia43. RAN translation may initiate at non-AUG codons via cap-dependent scanning mechanisms in multiple reading frames43–46; however, the products of RAN translation are very long repeat polypeptides, and to date no smORFs have been reported to undergo RAN translation.

Functions and regulation of sORFs that initiate with non-AUG start codons

Upstream open reading frames (uORFs) are a class of smORFs encoded in 5′ UTRs of mRNAs that function as cis-translational regulators in eukaryotic transcripts. AUG-initiating uORFs have been identified in up to 40% of human mRNAs, and generally exert a repressive effect on translation of the downstream, annotated protein-coding CDS due to inefficient reinitiation47. However, non-AUG-initiated uORFs are as common in some genomes as AUG-initiated uORFs16, and near-cognate initiation in the 5′ UTR has a more complex relationship to translation of the downstream gene. For example, during meiosis in yeast, near-cognate uORF translation was positively correlated with translation of the downstream cistron, whereas upstream AUG start codons were repressive28. A more recent study confirmed these findings; using a machine learning approach with a statistical control, 982 statistically significant uORFs were identified in 791 S. cerevisiae genes48. Examination of seven predictions with dual fluorescence reporter plasmid revealed four non-AUG and two AUG uORFs affected downstream coding sequence translation. Further genome-wide analysis revealed that genes with AUG uORFs had significantly lower translation efficiencies than genes without uORFs, while genes with non-AUG uORFs had higher translation efficiencies than genes without uORFs. These results are consistent with previous reports of upregulation of expression of EPRS and GADD45G during stress conditions49. The mechanism of uORF regulation during cellular stress has been recently reviewed21,50–52; briefly, cellular stress in eukaryotes induces phosphorylation of the eIF2α subunit of the eIF2 complex, inhibiting its ability to initiate translation of AUG-initiating uORFs. Under these conditions, relative activity of the alternative initiation factor eIF2A is increased; this complex specifically promotes translation from near-cognate start codons in the 5′ UTR as well as downstream canonical protein coding sequences.

Non-AUG uORF expression is also highly regulated and contributes to translational control during tumorigenesis. Ribosome profiling of the epidermis of wild-type and SOX2-expressing mice revealed that the translation of ORFs in the 5′ UTR is particularly differential upon oncogene induction, with a median increase of 1.84-fold53. Further pathway analysis revealed that these differential uORFs are co-encoded with downstream ORFs involved in mechanisms of cancer, stem-cell pluripotency and Wnt/β-catenin signaling, suggesting that during tumor initiation, the translational initiation apparatus is redirected towards uORFs of cancer-related mRNAs. Interestingly, the majority of these uORFs initiated with a CUG or GUG start codon and enhanced the translational efficiency of oncogenic mRNAs.

Non-AUG sORFs that exhibit regulated expression have also been observed in prokaryotes. Quantitative proteomics of E. coli K-12 revealed two sORFs, ymcF and ynfQ, that are specifically expressed during cold shock54. ymcF and ynfQ map to intergenic sequences downstream of the known cold shock proteins CspG and CspI, respectively, and are initiated from rare AUU start codons. These two sORFs are conserved in related Gram-negative bacteria, and are predicted to be structured. In a parallel study, a small, membrane-associated sORF, gndA, was identified during heat shock, which is encoded within the gndP gene in an alternative reading frame19. Later, an antibiotic-assisted ribosome profiling study revealed that gndA is initiated from a UUG start codon15. These non-AUG-initiated sORFs exhibit regulated expression under specific stress conditions, and, considering their conservation, predicted structure and/or subcellular localization, it is possible that the encoded small proteins are functional in trans; however, these functions have not yet been demonstrated.

Several sORFs that initiate at non-AUG start codons in mammalian cells have recently been demonstrated to be functional in disease and stress states. In one example, ribosome profiling of lipopolysaccharide (LPS)-treated mouse macrophages revealed 96 non-canonical smORFs are translated from lncRNAs, 55% of which initiated from non-AUG start codons. A smORF translated from lncRNA Aw112010, which initiates from a CUG start codon, was shown to play an important role in the response to bacterial infection. Disruption of translation of Aw112010 with a stop codon knock-in resulted in accelerated weight loss and higher bacterial burden in the liver and spleen of mice during S. Typhimurium infection, compared with wild-type litter-mates55. In another example, the mitochondrial ribosomal protein L18 (MRPL18) gene initiates translation from a downstream CUG start codon during heat shock. This alternative translation start site generates a truncated protein lacking the mitochondrial targeting signal, promoting localization of this N-terminaly truncated MRPL18 isoform to the cytoplasm instead of the mitochondria during heat shock. Truncated, cytosolic MRPL18 incorporates into the 80S ribosome and promotes synthesis of heat shock proteins during stress.56

Perspective

It is now clear that non-AUG-initiated translation is pervasive and conserved from bacteria to human, and is enriched among sORFs relative to main ORFs. Several functions for translation of sORFs from non-AUG start codons have been proposed. First, uORFs, or sORFs in the 5′ UTR, generally switch from negative to positive translational regulatory roles when they initiate with near cognate, rather than AUG, start codons, especially under developmental and/or stress conditions. Secondly, sORFs that function in trans can exhibit regulated translation under stress conditions or other stimuli. It is likely that the start codon identity is involved in tuning the expression level and also condition-specific translational regulation of all of these classes of sORFs.

It is also possible that ubiquitous non-AUG initiation of sORFs has an evolutionary origin. It has been proposed that sORFs represent instances of de novo gene birth, in which non-genic smORFs are transcribed and translated at low levels, occasionally acquiring adaptive mutations13. This model is consistent with the intermediate conservation of sORFs3,13, and would also be consistent with enrichment of non-AUG start codons among protogenes, which arise randomly from the genomic sequence and have not yet been selected for optimal57, AUG-driven translation.

Moving forward, only a handful of non-AUG translation events have been characterized in molecular detail, and non-AUG-mediated translation has been globally profiled under only a few conditions. It is possible that under additional cellular and disease conditions, other non-AUG-initiated ORFs that exhibit specific expression may remain to be identified. Further investigation of the regulation and functions of these sORFs will aid their identification and characterization. Taken together, the preponderance of non-AUG start codons driving sORF expression suggests that our model of translation initiation, and our understanding of the mechanism by which information is encoded in genomes, must be revised.

Acknowledgments.

This work was supported in part by the Searle Scholars Program, a Smith Family Foundation Odyssey Award, the NIH (R01GM122984), and Yale University West Campus start-up funds (to S.A.S.). X. C. was supported in part by a Rudolph J. Anderson postdoctoral fellowship from Yale University.

Footnotes

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