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The regulatory potential of upstream open reading frames in eukaryotic gene expression Klaus Wethmar1,2∗ Upstream open reading frames (uORFs) are prevalent cis-regulatory sequence elements in the transcript leader sequences (TLSs) of eukaryotic mRNAs. The majority of uORFs is considered to repress downstream translation by the consumption of functional pre-initiation complexes or by inhibiting unrestrained progression of the ribosome. Under distinct conditions, specific uORF properties or sequential arrangements of uORFs can oppositely confer enhanced translation of the main coding sequence, designating uORFs as versatile modifiers of gene expression. Ribosome profiling and proteomic studies demonstrated widespread translational activity at AUG- and non-AUG-initiated uORFs in eukaryotic transcriptomes from yeast to human and several reports linked defective uORF-mediated translational control to the development of human diseases. This review summarizes the structural features affecting uORF-mediated translational control in eukaryotes and describes the highly divergent mechanisms of uORF regulation that result in repression or induction of downstream protein translation. © 2014 John Wiley & Sons, Ltd.

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WIREs RNA 2014. doi: 10.1002/wrna.1245

INTRODUCTION

T

ranslational initiation within the transcript leader sequence (TLS)a gives rise to an upstream open reading frame (uORF) that either overlaps or terminates before the initiation codon of the main protein-coding sequence (CDS). Upstream ORFs may serve as rapid response elements, allowing cells to immediately adopt protein production to altered environmental conditions at the level of translation. Computational sequence analyses identified uORFs in 13, 44, and 49% of yeast, mouse, and human transcripts, respectively.1,2 Validating these observations, upstream translational initiation sites (uTISs) were recently detected in >50% of human transcripts by ribosome profiling, suggesting a widespread role ∗ Correspondence

to: [email protected]

1 Max-Delbrueck-Center 2 Helios

for Molecular Medicine, Berlin, Germany Klinikum Berlin-Buch, Berlin, Germany

Conflict of interest: The author has declared no conflicts of interest for this article.

of uORFs in gene expression regulation.3 Interestingly, only about a quarter of upstream translation appeared to be initiated by AUG codons, while more than 70% of initiation was observed at near-cognate start codons that differ in one base from the classical AUG triplet. About 64–85% of experimentally identified uTISs were conserved between human and mouse, indicating a functional role for these elements.3 Nevertheless, while the overall prevalence of uAUGs is lower than could be expected by chance (especially for those uAUGs that may initiate CDS-overlapping uORFs4 ), AUG is the nucleotide triplet best conserved between human and mouse in the TLS.5 Both observations argue for a functional role of conserved uORFs and a selective evolutionary loss of nonfunctional uORF start sites. The vast majority of investigations up to now focused on the analysis of uAUG-initiated uORFs: At present, approximately 100 human, 50 murine, and a total of 110 transcripts of other eukaryotic or viral taxa have been experimentally analyzed.6 These studies revealed a multitude of structural and functional properties that determine the regulatory impact

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of uORFs on messenger RNA (mRNA) translation. In general, the regulatory potential of uORFs is easily comprehensible by applying the scanning model of cap-dependent translational initiation7,8 : Upon engagement of the small ribosomal subunit with a transcript via the 5′ -located 7-methyl-guanosine (m7G) mRNA cap structure, a 43S pre-initiation complex is assembled, consisting of the 40S ribosomal subunit, the eIF2–GTP–Met-tRNAi ternary complex (eIF-TC), and additional eukaryotic initiation factors.7,9 The pre-initiation complex scans the mRNA toward the 3′ -end until the Met-tRNAi anti-codon matches a functional initiation codon. Joining with the 60S ribosomal subunit generates a fully functional ribosome and permits initiation of translation. Consequently, translational initiation at a uORF start site results in consumption of a functional pre-initiation complex and is generally associated with reduced capacity of downstream translational initiation. Nevertheless, despite of the simplicity of the aforementioned model, uORFs do not always repress translation of the CDS, as specific structural features of individual mRNAs or a certain arrangement of subsequent uORFs may permit induction of translation at downstream initiation codons. In fact, due to highly variable transcript-specific structural properties and a plethora of resulting mechanistic modes and functional outcomes, uORF-mediated translational control is anything but uniform and far from being understood. This review summarizes current knowledge about uORF-mediated translational control mechanisms in eukaryotes. It is based on a comprehensive literature database on eukaryotic uORF biology, uORFdb,6 which contains categorized and indexed entries for all uORF-related publications listed in NCBI’s PubMed. To allow a systematic description of the complex field of uORF biology, the review will summarize the structural properties that affect uORF functions and describe the multiple functional mechanisms uORFs apply to confer gene expression regulation at the translational level. Finally, the review will briefly outline the documented and the potential medical implications of defective uORF-mediated translational control and delineate some open questions and future prospects of the field.

STRUCTURAL PROPERTIES AFFECTING THE REGULATORY POTENTIAL OF uORFs The structural diversity of uORFs is immense and each uORF functions in a transcript-specific context (Figure 1). The regulatory capacity of a uORF is

(a) Fully upstream

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FIGURE 1 | Structural properties affecting the regulatory potential of upstream open reading frames (uORFs). (a) The models depict various structural appearances of uORFs (orange/red bars). Transcript leader sequences (black lines) may contain single or multiple uORFs positioned between the 5′ cap structure (black circle) and the CDS (blue bars). Upstream ORFs may be located fully upstream, overlap the CDS initiation codon or may be interlaced between alternative initiation codons within the CDS. Green arrows indicate the two possible ways of CDS translation in the presence of uORFs, i.e., reinitiation and/or leaky scanning. Upstream ORFs may overlap each other in- or out-of-frame, or appear as a group of multiple individual uORFs. (b) The regulatory function of a uORF is strongly affected by numerous individual features including its length and position within the TLS, the quality of the Kozak consensus sequence surrounding the initiation codon (green line), the composition of the termination context surrounding the upstream stop codon (red line), and by stable secondary structures (black loops) located elsewhere in the messenger RNA (mRNA).

greatly dependent on its position within the TLS, the surrounding secondary and tertiary structures of the mRNA, and in some cases on specific features of its peptide or mRNA sequence. Furthermore, the sequence context surrounding the uORF initiation codon is of outstanding importance, as it

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contributes substantially to the efficiency of initiation at the uORF start codon—a prerequisite for every further regulatory impact of a uORF. In some cases, the termination context crucially affects uORF function by determining the reinitiation capacity of post-termination ribosomes. Finally, trans-acting factors, including signaling effectors, metabolites or small molecule interactors, co-regulate uORF functions, yet, are always dependent on the entirety of all other structural features.

Alternative Promoter Usage, Alternative Splicing, and Tissue Specificity The presence or absence of uORFs in transcript variants derived from the same gene fundamentally alters the regulatory framework of translational control. Transcript-specific gain or loss-of-uORFs may occur due to alternative promoter usage or alternative splicing in specific cell types, tissues or in association with specific diseases. For example, the oncogene MDM2 is transcribed in a long (L-MDM2) and a short (S-MDM2) isoform from alternative promoters.10 While the long isoform contains two uORFs that synergistically repress translation approximately 10-fold,11 tumor specific expression of the S-MDM2 transcript was associated with elevated translation of MDM2 and subsequent inhibition of p53-mediated apoptosis.10 Similarly, transcription of the human estrogen receptor 2 (ESR2) gene from distinct promoters generated transcripts containing variable numbers of uORFs that acted in a cell type-specific fashion and were implicated in carcinogenesis.12,13 Other examples of transcript variant-dependent presence of uORFs and altered downstream translation have been described for human estrogen receptor 1,14 testes-specific expression of a long transcript variant of mouse peroxiredoxin V,15 and human GRB2-related adaptor protein 2.16 The latter is translationally silenced by uORFs contained in a megacaryocyte-specific transcript. Interestingly, thrombin signaling in activated platelets overruled uORF-mediated silencing, yet the exact regulatory mechanism has not yet been elucidated. Several more general investigations have been performed to characterize the contribution of uORFs to translational gene regulation: When yeast was grown under 18 different environmental conditions, differential splicing or transcriptional start site selection altered the presence or absence of uORFs in 431 cases, suggesting a general impact of TLS diversity on gene regulation.17 Another recent study evaluated the role of mammalian pre-mRNA splicing on the

distribution of alternative transcripts to cytoplasmic versus polysomal fractions and suggested that the presence or absence of uORFs contributed substantially to the amount of polysomal association.18 In mice, a systematic analysis of five connexin genes in 17 distinct tissues revealed a high degree of tissue-specific promoter usage associated with the generation of alternative TLS variants. The results suggested that both, transcriptional and translational regulation, are implicated in cell type-specific expression of this important group of developmental genes.19 Similarly, part of the translational inhibition of many ubiquitously expressed genes in mouse somatic spermatogenic cells was attributed to the cell type-specific usage of alternative transcriptional start sites, generating uORF containing transcript variants.20

Secondary Structures of the mRNA Stable secondary structures of the mRNA can affect the translation of uORFs and of the main CDS depending on their sizes, positions, and energetic stabilities. Translational initiation requires local unwinding of the mRNA, which involves recruitment and activation of the ATP-dependent helicase eIF4A.7,21 Hairpin structures are often observed in G/C-rich parts of the TLS and are most inhibitory to translational initiation when positioned close to the 5′ -cap structure by preventing ribosome engagement with the RNA.22,23 On the other hand, stable secondary structures in a defined position downstream of a uORF may prevent leaky scanning across its initiation codon by slowing down scanning ribosomes. Longer dwell time may permit translation of the uORF and inhibit subsequent translation of the CDS.24,25 Along these lines, recent computational data suggest that stable secondary and tertiary structures play an important role in facilitating initiation at non-AUG codons,26 yet, these observations require further experimental validation. In contrast to inhibitory downstream effects of uORF-related hairpins, reinitiation promoting elements (RPE) have been identified in the surroundings of uORFs in yeast GCN4 and YAP1.27 These elements were shown to stabilize post-termination 40S ribosomal subunits to resume scanning upon uORF translation via an interaction with eIF3A/TIF32.

The Kozak Consensus Sequence Translational initiation is a prerequisite for the regulatory function of a uORF. As we have learned from the classical permutation experiments performed by

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Kozak8,28,29 and from more recent computational sequence analyses,4,30 the nucleotide context surrounding an initiation codon strongly affects the translational initiation efficiency in mammalian cells. The optimal surrounding sequence for ribosome initiation is GCCRCCAUGG, with R representing a purine base. The nucleotides at −3 and +4 of the so-called Kozak consensus sequence are of particular regulatory importance. Consequently, the Kozak context is classified as strong (both crucial bases match the consensus), adequate/intermediate (one of the crucial bases matches the consensus), or weak (no consensus match at −3 and +4). Nevertheless, the Kozak context alone does not independently predict the frequency of initiation or the regulatory impact of a uORF, as many more transcript-specific parameters affect uORF function.

35 and personal observations), no positive correlation between uORF overlap and the degree of CDS repression could be verified for naturally occurring overlap-uORFs in the above-mentioned association study.2 As so often in uORF biology, attempts to establish general rules from individual observations in these latter cases failed, most likely due to the high number of additional structural features affecting uORF-mediated translational control. Despite the controversies, the deduced model, where essential ribosomal (re)initiation factors gradually dissociate from uORF-elongating ribosomes over time and need to be re-loaded to facilitate downstream reinitiation may be true for many transcripts.8

The Termination Context Positioning of uORFs Within the TLS Multiple experiments demonstrated that altering the position of uORFs within the TLS strongly affects the regulatory potential on downstream translation. Individual transcript analyses and a large-scale correlation study confirmed a positive correlation between uORF-mediated repression of the CDS and (I) the number of uORFs per transcript, (II) the distance from the 5′ -cap to the uORF initiation codon, (III) the degree of evolutionary conservation of the uORF initiation codon, and (IV) the quality of the uORF-related Kozak consensus sequence.2 Furthermore, a number of individual studies suggested other structural properties as important for uORF regulation. The degree of downstream translational repression was shown to increase with the length of the preceding uORF, as elongating ribosomes may gradually shed crucial co-factors required for efficient reinitiation.31,32 Vice versa, experiments using yeast GCN4 or artificial constructs showed that lengthening of the inter-cistronic distance between the uORF stop codon and the downstream CDS relieved downstream translational repression.24,33 Here, the assumption was that longer distances between uStop and CDS-start codons would provide more time for reformation of a functional pre-initiation complex. Nevertheless, such effects were neither reproduced in other transcripts, e.g., CEBPA and -B,34 nor validated more generally in an unbiased comprehensive association study correlating uORF features and protein abundance.2 Similarly, although experimental transformation of a uORF located fully upstream into a uORF overlapping the CDS most frequently results in enhanced repression of CDS translation (Ref

While translation initiation is a prerequisite of uORF-mediated translational control, ribosomal termination at uORF stop codons also appears to be of great importance. The nucleotide context surrounding uORF termination codons can contribute to termination-induced ribosomal pausing, stalling and ultimately to mRNA decay. Furthermore, specific features of the termination context can leave the ribosome with variable capacity for reinitiation, as first shown for a 10-bp element downstream of the fourth uORF of yeast GCN4.36,37 Mutational experiments showed that the reinitiation-inhibitory effect of the uORF4 termination context was transferable to other uORFs and could be overcome by introducing A/U-rich codons to its own sequence or to the pre-terminal codon of the preceding uORF. This suggested a model, where stable base pairing of nucleotides surrounding the uORF termination codon with the translational machinery itself or with other sequences in the GCN4 transcript accounted for the reinitiation-inhibitory effect of uORF4. In another case, the G/C-rich termination context of yeast CAD1 functioned as a transferable reinitiation-inhibitory element by promoting UPF1-independent mRNA degradation upon post-termination ribosomal release.38

MECHANISTIC MODES OF uORF-MEDIATED TRANSLATIONAL CONTROL Data obtained from individual analyses of more than 250 eukaryotic transcripts uncovered multiple distinct mechanisms of uORF-mediated translational control

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(for a bullet point summary see Box 1). The ultimate impact on gene regulation is always a function of such independent or integrated regulatory effects, rendering every uORF truly individual. The subsequent paragraphs provide an overview of the highly divergent functional modes of uORF-mediated gene regulation. BOX 1 MODES OF uORF-MEDIATED TRANSLATIONAL CONTROL • Tissue- and transcript-specific presence of uORFs due to alternative splicing and alternative promoter usage. • Integration of global translational conditions by specific arrangements of subsequent uORFs. • Consumption and condition-dependent reconstitution of functional pre-initiation complexes. • Conversion of scanning ribosomes into a ‘reinitiation mode’. • Induction of ribosome stalling by • Specific interactions between the nascent uORF peptide, a small molecule interactor and the translating ribosome. • Ligand-independent inhibition of ribosome progression by direct interaction between specific uORF-peptide or -nucleotide sequences and the translating ribosome. • Disturbed termination due to inhibitory post-termination sequences. • Induction of nonsense-mediated decay by • Ribosome pausing at uORF-contained nonsense codons. • ‘Premature’ ribosomal termination at uORF stop codons upstream of the CDS. • Start site selection in transcripts with alternative downstream translational initiation codons. • Shunting of ribosomes across inhibitory secondary structures. • Co-factor dependent translational regulation.

Integration of Global Translational Conditions Translational control mechanisms are widely considered to implement rapid cellular adjustments to

changing environmental conditions. In this line, uORF-mediated translational regulation was implicated in the regulatory response program toward various cellular stresses, including nutrient starvation, unfolded protein response, oxidative stress and others.39 According to the current model of cap-dependent translation, each initiation event consumes a functional ternary pre-initiation complex.7 This implies the requirement of its reconstitution in order to facilitate subsequent reinitiation events. A number of crucial eukaryotic initiation factors (eIFs) disengage from the ribosome upon 60S subunit joining at the initiation step and it has been proposed that additional co-factors are depleted in a dynamic manner during the elongation phase.31,40–42 Several lines of evidence indicate an important role for eIF3 and eIF4G whose persistent interaction with the 40S subunit may allow sustained scanning and efficient reloading of post-termination ribosomes.42 The model of co-factor reloading is widely built on observations from the analysis of the TLS of yeast transcription factor GCN4, where four uORFs mediate the paradoxical induction of GCN4 translation under reduced global translational conditions.43–46 The translational status of a cell is ultimately defined by the availability of indispensable co-factors required for translational initiation and elongation. A central component of stress signal-mediated translational control is eukaryotic translation initiation factor 2A (eIF2A) that becomes phosphorylated at S51 upon stress kinase signaling via, e.g., GCN2 or PERK. In its phosphorylated form eIF2A cannot serve to reconstitute the eIF2–GTP–Met-tRNAi complex required to reconstitute a functional pre-initiation complex. In yeast GCN4, high levels of nonphosphorylated eIF2A under favorable translational conditions permit rapid reloading of ribosomes that translated uORF1. These ribosomes reinitiate at inhibitory uORFs 2–4, preventing translation of the GCN4 main protein-coding region (Figure 2(a)). Under starving conditions, reloading is delayed and ribosomes leaky scan across uORFs 2–4. Only upon prolonged scanning toward the coding region, ribosomes regain initiation competence and are primed to translate GCN4 protein.45 The first mammalian transcript resembling this regulatory pathway was mouse activating transcription factor 4 (Atf4).39 Drug-induced PERK and GCN2 signaling resulted in translational de-repression Atf4 protein production and facilitated transcriptional activation of downstream targets implicated in amino acid biosynthesis. Subsequently, additional examples of eIF2A-dependent translational control mechanisms

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have been described for ATF5,47 CCAAT/enhancer binding protein A and -B,35 and the glycoprotein receptor CD36.48

(a) Integration of global translational conditions High

elF2 GTP Met-tRNA

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(E) Ribosome shunting Inhibitory secondary structure

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FIGURE 2 | Mechanistic modes of uORF-mediated translational control. (a) Simplified model of uORF-mediated expression regulation of yeast GCN4 in response to stress-induced depletion of the eIF2–GTP–Met-tRNAi complex. See main text for a detailed description. The figure uses the same graphical layout as introduced in Figure 1. (b–e) Graphic representations of ligand-induced ribosome stalling at peptide-specific uORFs, of nonsense-mediated decay induced by ribosome stalling at uORF termination codons, of uORF-directed start site selection of post-termination ribosomes, and of uORF-dependent ribosome shunting across inhibitory secondary structures within the TLS.

Ribosome Stalling Specialized uORFs in plants, yeast, and mammals encode peptides that mediate adequate adjustments of protein translation in response to specific metabolites. Human AMD1, an important intermediate enzyme in polyamine biosynthesis, encodes a hexapeptide (MAGDIS) in its uORF that was shown to repress translation by a cis-regulatory and cell type-specific ribosome stalling mechanism.49,50 High levels of polyamines induce a stable interaction between the translating ribosome and the nascent uORF peptide, preventing proper termination and resulting in the induction of mRNA decay pathways. The regulation of AMD1 expression by uORF-mediated integration of polyamine levels is conserved in plants and mammals.51 Similarly, polyamines also regulate the translation rates of ornithine decarboxylase genes via a uORF conserved from yeast to mammals.52 Another example of metabolite-mediated uORF regulation comes from the small subunit of arginine-specific carbamoyl-phosphate synthetase (CPS-A). Ortholog genes in yeast and Neurospora crassa, CPA1 and arg-2, encode uORF-peptides that sense cellular arginine levels and induce ribosome stalling at the uORF termination codon upon arginine binding.53,54 Stalled ribosomes are not only inhibitory to ribosomal progression toward the CDS, but also induce nonsense-mediated decay (NMD) of CPA1 mRNA.55 Structural insights to the process of arginine induced ribosomal stalling have been provided.56 Furthermore, the Arabidopsis thaliana transcription factors GBF6 and bZIP2 contain uORF-encoded sucrose-control peptides, conferring translational regulation through the integration of sucrose levels.57 Independent of small molecule regulators, a direct interaction between the uORF peptide chain and the translating ribosome can inhibit proper ribosomal progression or termination. The termination inhibitory uORF peptide of glycoprotein 48 (gp48) of human herpes virus 5 (HHV-5) induces ribosomal stalling by preventing the hydrolysis of the peptidyl-tRNA bond between the nascent peptide and the tRNA decoding the C-terminal uORF codon, leaving behind a detectable peptidyl-tRNA-compound.58 In other cases including human 𝛽 2 adrenergic receptor (ADRB2),59 mouse retinoic acid receptor 𝛽 (Rarb),60

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DDIT3,61 and MDM211 translational repression was also dependent on a specific uORF peptide or mRNA sequence, but the exact inhibitory mechanism remained unexplored. An exceptional case of uORF-peptide-regulation was found in plants, where the uORF peptide of an alternatively spliced HAP2 transcript in Medicago truncatula acted in trans to repress HAP2 expression.62

NMD and RNA Destabilization NMD serves as a cellular surveillance mechanism to deplete transcripts that show signs of premature translational termination.63 Premature termination or ribosome stalling may occur at nonsense codons derived by mutations, incorrect splicing, or aberrant initiation site selection. Genome-wide studies in mammalian cells,64 Caenorhabditis elegans65 and yeast4,66 demonstrated that uORF-bearing transcripts are particular susceptible to targeted degradation by NMD, attributed to the 5′ -near termination events at uORF stop codons. In addition, another mechanism of uORFmediated mRNA destabilization has been reported in yeast that involves the release of post-termination ribosomes from the TLS.38

Induction of Downstream Translation and Start Site Selection Enhancement of downstream protein translation through uORF regulation mostly involves an indirect stimulatory effect arising from the neglect or bypass of downstream inhibitory structures or sequence elements. As outlined above, the paradox induction of human DDIT3,67 mouse Atf4,68 and yeast GCN445 upon cellular stress signals results from delayed reloading of post-termination ribosomes and the associated leaky scanning across downstream uORF start codons. Another example of uORF-mediated induction of protein translation is human CD36, an important glycoprotein receptor implicated in atherosclerosis. High glucose levels were found to increase translation of CD36 by enhancing the reinitiation rate of ribosomes that translated the CD36 uORF.48 Glucose-mediated translational induction of CD36 was validated in primary human macrophages and vascular lesions, implying a mechanism for accelerated atherosclerosis in patients suffering from diabetes. Only few exceptions from the generally indirect nature of uORF-mediated translational induction were described. For mouse mitochondrial transporter protein Ucp2, addition of glutamine was shown to

promote translation of the CDS in a uORF-dependent fashion, yet the exact mechanism of the phenomenon remained unclear.69 In another case, translation of a uORF in the mouse Slc7a1 transcript induced unfolding of an inhibitory structure within the TLS and activated a downstream IRES element, resulting in enhanced translation of the CDS.70 Furthermore, skipping of the uORF-proximal TIS can result in increased translation of N-terminally truncated protein isoforms from alternative downstream initiation codons. This was studied comprehensively in the mammalian transcription factors CCAAT/enhancer binding protein 𝛼 and -𝛽 (CEBPA and -B), where uORF-mediated translational control induced the alternative translation of short protein isoforms that act in an auto-inhibitory way with respect to the longer CEBP protein versions.35 The balanced expression of CEBPA and -B isoforms proved to be important in myeloid cell fate decisions, liver regeneration, and in the development of acute myeloid leukemia.71–73 Other examples of uORF-controlled isoform production include the hematopoietic and proto-oncogenic transcription factors TAL174 and FLI1.75

Shunting Upstream ORF-dependent ribosome shunting across highly structured and inhibitory parts of the TLS was first observed and mechanistically characterized in plant Cauliflower mosaic viruses (CaMV).76,77 Here, shunting involves translation of a uORF and a defined landing sequence 3′ -prime of an inhibitory secondary structure within the TLS. Further experiments using synthetic mRNA leader sequences demonstrated that shunting not only requires complete translation and termination of a uORF, but is also dependent on a permissive post-termination sequence context allowing ribosomal reinitiation.78 A similar ribosomal shunting mechanism was identified in the viral pre-genomic RNA in Rice tungro bacilliform virus and Rice tungro spherical virus.79 To date, the only human/vertebrate example for uORF-mediated shunting was described for anti-apoptotic BIRC3, where a highly structured part of the TLS is bypassed in a stress dependent fashion.80

Co-Regulation by RNA-Binding Proteins The uORF of Drosophila msl-2 is currently the only example for a uORF being co-regulated by a defined protein factor. A specific RNA-motif, located 27–35 bp downstream of the uORF termination codon in various Drosophila species, facilitated binding of the co-factor protein SXL that mediated enhanced

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initiation at the uORF start site and thereby caused repression of the msl-2 CDS.81

RECENT ADVANCES IN METHODOLOGY TO STUDY uORF BIOLOGY Ribosome Profiling Ribosome profiling, i.e., deep sequencing of ribosome-protected RNA fragments, has revolutionized the analysis of translational control mechanisms, as it combined the precision of nucleotide specific signals with the global informative value of a transcriptome-wide analysis.82 Ribosome profiles uncovered extensive translational changes upon induction of cellular differentiation or in response to stress conditions including nutrient starvation, hydrogen peroxide, or drug treatment.83–85 Inhibition of mTOR signaling in a prostate cancer cell line identified a subset of translationally regulated genes involved in cancer invasion and metastasis.85 Ribosome profiling further revealed that the 5′ terminal oligopyrimidine motif is the unifying mRNA feature that confers mTORC1-dependent translational control to the respective mRNAs.86 In general, stress conditions shifted translational activity toward noncanonical initiation sites, resulting in increased translation of uORFs, as well as extended and truncated versions of annotated proteins in yeast, mouse, and human transcriptomes.3,82 Surprisingly, the most frequently detected translational initiation site within the TLS was CUG, followed by AUG and a number of other near-cognate initiation codons. As AUG uORFs were shown to be most effective in repressing translation at the downstream CDS,3 it remains to be experimentally determined to what extent non-AUG codons serve as functional uTISs in vivo. Computational simulation suggested that steric blocks imposed by downstream secondary and tertiary structures of the mRNA molecule may contribute to noncanonical start site selection by scanning ribosomes.26 Currently, human BIRC2 and yeast GCN4 are the only two examples where translational activity at non-AUG uORFs has been functionally analyzed in individual experimental settings.87,88 While the BIRC2 uORF showed strong regulatory impact on BIRC2 translation, the translated non-AUG uORF of GCN4 appeared to be dispensable for proper translational regulation of the CDS.

Animal Models While most functional data on uORF-mediated translational control has been deduced from reporter

gene studies, ribosome association experiments or by monitoring RNA decay, two prospective animal models have been established to experimentally analyze uORF functions in the living animal. First, a point mutation designed to abolish initiation at the uORF start codon of CCAAT/enhancer binding protein 𝛽 (CEBPB) was introduced in mice.72 The loss-of-uORF mutation resulted in reduced expression of the truncated, auto-inhibitory isoform of CEBPB, validating previous results obtained from cell culture experiments.35 Phenotypically, mutant mice displayed defects in liver regeneration, osteoclast differentiation and showed altered expression of multiple cell cycle regulatory genes. Second, a zebrafish transgenic reporter line was generated that contained the stress-responsive uORF of human CCAAT/enhancer binding protein homologous protein (CEBPZ/DDIT3) preceding a GFP repoter gene.89 While the uORF silenced GFP expression under steady state conditions, heat shock, and endoplasmic reticulum stresses resulted in de-repressed translation of the reporter gene in certain tissues, suggesting cell type specificity of DDIT3-uORF translational regulation. In a third retrospective case, the pathogenic cause for the phenotype of pre-existing hairpoor and near-naked mouse models could be traced back to loss-of-uORF mutations in the initiation codon of the second Hr uORF, as reported earlier for human Marie Unna hereditary hypotrichosis.90,91

Proteomics The first proteomic identification of uORF-encoded peptides was achieved about ten years ago, when Oyama and colleagues identified 54 small uORF-derived polypeptides of less than 100 amino acids from lysates of the human K562 cell line.92 A more recent study detected a total of 90 short ORF-encoded polypeptides (SEPs) and revealed that 15 of the SEPs were derived from uORFs while the remaining peptides arose from other parts of mRNAs or from noncoding RNAs.93 Validating ribosomal profiling data, a substantial fraction of these SEPs were generated from non-AUG initiation codons, suggesting that alternative translational initiation is more frequent than previously anticipated. Overall, even in the latest report of a mass-spectrometric analysis that applied a refined peptide library based on ribosomal profiling data, the number of detected uORF-peptides was surprisingly small.94 As discussed in the latter publication, uORF-peptides may rapidly be degraded due to their small size or low abundance, or may simply not be detected due to their frequently nontryptic nature

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or loss in enrichment protocols. Thus, it remains to be determined individually, how many of the approximately 200 human uORFs that were suggested to encode functional peptides based on the high degree of amino acid sequence conservation between mouse and human, are actually translated in vivo.95

CONCLUSION Numerous individual reports and a growing list of ribosome profiles and proteomic studies documented high translational activity at uORFs and other alternative initiation codons, demonstrating that eukaryotic proteomes are much more diverse than reflected by the current annotations in public databases. In this context, a central and still unresolved question related to uORF biology is: What discriminates a coincidental start codon, derived by random mutations or stochastic shuffling of the four alternative nucleotides, from a start codon that serves as a functional uORF with extensive regulatory potential? Todays sequence analyzing tools such as sORF finder96 or uPEPperoni97 may help to identify potential candidates, but fail to reliably predict functional alternative TISs. As individual experimental testing is not an option, combined efforts of refined ribosome profiling strategies, uORF-adapted mass-spec enrichment protocols and unbiased proteogenomic approaches are required to further separate the wheat from the chaff. Another open task is to investigate whether adjacent or distal molecular interaction motives co-regulate uORFs in a similar fashion as observed for the msl-2/SXL interaction in Drosophila.81 Furthermore, the ability of specific uORFs to sense nutrient supplies or the concentrations of other small molecule-interactors encourage systematic screens for additional co-regulatory molecules. Identification of such protein- or small molecule-interactors would render uORFs targetable for medical application and may ultimately permit drug-mediated expression control of genes involved in the pathogenesis of specific diseases. A step in this direction has recently been taken by a comprehensive screening approach that identified multiple interactions between short RNA motifs and small molecular compounds.98 At present, several diseases have been traced back to genetic changes altering uORF-mediated control (see Box 2). In the light of the abundant prevalence of uORFs and the high uORF-related translational activity identified by ribosome profiling, it appears likely that gene products acting in

BOX 2 CLINICAL ASSOCIATIONS OF uORF-ALTERING SNPs AND ACQUIRED GENETIC LESIONS Single nucleotide polymorphisms (SNPs) and acquired genetic lesions affecting the initiation codon or the coding sequence of a uORF are highly prevalent in the human genome.2,6,99 The resulting variations in genotype-specific translational control may contribute to phenotypic divergence and may aggravate the susceptibility to specific diseases. Strongest evidence for a pathogenetic role of defective uORF-mediated translational control was derived from the analyses of four inherited diseases: (1) Hereditary thrombocytosis was shown to result from a splice donor site mutation causing excision of exon3 together with an inhibitory uORF from the TLS of the thrombopoietin gene (THPO). Loss of the inhibitory uORF de-repressed translation of THPO protein and resulted in enhanced platelet production.100 (2) A germline mutation introducing a uORF start site in the TLS of cyclin-dependent kinase inhibitor 2A (CDKN2A) caused reduced expression of this cell cycle inhibitory protein and was the first noncoding mutation in CDKN2A associated with the development of hereditary melanoma.101 (3) Several abolishing mutations within the second uORF of hairless homolog (U2HR), found in a number of Chinese and middle east families, caused increased expression of HR protein and resulted in the development of Marie Unna hereditary hair loss.91,102,103 Curiously, a mutation in U2HR could also be detected in a descendant of the affected family described in the original report of 1925.104 (4) More recently, a 4-bp deletion within the uORF of the CDKN1B TLS was identified in a patient suffering from multiple endocrine neoplasia type 4 (MEN4) syndrome.105 This deletion caused a profound lengthening of the uORF and an equivalent shortening of the uStop-to-CDS distance, resulting in pathogenic reduction of CDKN1B expression. Other uORF-related genotype–phenotype associations, implicating such ubiquitous diseases as Diabetes, Alzheimer’s syndrome, or atherosclerosis, have recently been summarized and reviewed elsewhere.2,6,99

a dose-dependent fashion (e.g., transcription factors, surface receptors, and tyrosine kinases) may be targets of as yet undiscovered pathogenic uORF mutations.

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The number of cases described today may underestimate the contribution of defective uORF functions in human diseases, as the majority of previous resequencing strategies excluded the analysis of the TLS by focusing on the coding exome. Thus, the escalating application of whole genome sequencing in cancer and other diseases has great potential to uncover additional uORF mutations with pathophysiologic importance in the near future.

NOTE a

Due to increasing evidence for AUG- and non-AUG-initiated translation within the 5′ -regions of eukaryotic transcripts, the widely used misnomer of the 5′ -untranslated region (5′ -UTR) is omitted. Instead, the term TLS is applied to describe any mRNA sequence preceding the initiation codon of the annotated main coding sequence.

ACKNOWLEDGMENTS I thank Achim Leutz for critical reading of the manuscript and numerous valuable advices along the various projects I pursued in his lab. This work was supported by the Deutsche Krebshilfe e.V., Bonn, Germany (grant 110525 to KW).

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