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Non-AUG Translation: a New Start for Protein Synthesis in Eukaryotes

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Non-AUG Translation: a New Start for Protein Synthesis in Eukaryotes Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Non-AUG translation: a new start for protein synthesis in eukaryotes Michael G. Kearse and Jeremy E. Wilusz Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, 19104 USA Although it was long thought that eukaryotic translation cognate codons that differ from AUG by only one nucleo- almost always initiates at an AUG start codon, recent ad- tide (e.g., CUG, GUG, and UUG) are used. vancements in ribosome footprint mapping have revealed Given the strong evolutionary pressure that is observed that non-AUG start codons are used at an astonishing fre- across all organisms to use AUG start codons, it may ap- quency. These non-AUG initiation events are not simply pear at first glance that initiation events from non-AUG errors but instead are used to generate or regulate proteins codons represent intrinsic errors of the translation ma- with key cellular functions; for example, during develop- chinery. This idea is directly challenged by the fact that ment or stress. Misregulation of non-AUG initiation a number of endogenous and viral proteins with important events contributes to multiple human diseases, including functions are derived solely from non-AUG start codons cancer and neurodegeneration, and modulation of non- (Curran and Kolakofsky 1988; Dorn et al. 1990; Xiao AUG usage may represent a novel therapeutic strategy. et al. 1991; Chang and Wang 2004; Tang et al. 2004; Beer- It is thus becoming increasingly clear that start codon se- man and Jongens 2011; Ivanov et al. 2011). For example, lection is regulated by many trans-acting initiation fac- DAP5 (also called eukaryotic initiation factor 4G2 tors as well as sequence/structural elements within [eIF4G2] or NAT1) plays a critical role in internal ribo- messenger RNAs and that non-AUG translation has a pro- some entry site (IRES)-mediated translation and is initiat- found impact on cellular states. ed solely from a GUG start codon in mouse and human cells (Imataka et al. 1997; Takahashi et al. 2005; Lewis et al. 2008; Marash et al. 2008; Liberman et al. 2015). Like- Eukaryotic genomes encode thousands of proteins with wise, in yeast, UUG and ACG start codons are used to ini- important structural and regulatory roles, and a signifi- tiate translation of the GRS1 and ALA1 transfer RNA cant amount of cellular energy is dedicated to transcrip- (tRNA) synthetases, respectively (Chang and Wang tion of messenger RNAs (mRNAs) and their subsequent 2004; Tang et al. 2004). These non-AUG translation translation into protein. Errors in translation can result events represent only the tip of the iceberg, as ribosome in wasteful production of inactive or deleterious proteins profiling has recently revealed thousands of novel initia- that misfold, aggregate, lack regulation, or otherwise dis- tion events at non-AUG codons (Ingolia et al. 2009, rupt cellular fitness (Drummond and Wilke 2009). It is 2011). Interestingly, not all near-cognate start codons are thus critical that ribosomes initiate at the appropriate co- used with equal efficiency, with CUG generally being don, incorporate the appropriate amino acids into the most efficient, followed by GUG, ACG, and AUU (Table growing polypeptide chain, and terminate only at the ap- 1). It should be noted that there is significant variation propriate stop codon (Zaher and Green 2009; Rozov in these efficiency measurements across assays. This is et al. 2016). Protein-coding sequences have traditionally likely because in vitro translation assays are strongly in- been defined as uninterrupted ORFs that begin with the fluenced by how the lysates are prepared (e.g., those pre- universal AUG start codon and end with one of three pared by gel filtration may lack low-molecular-weight stop codons (UAA, UGA, and UAG). However, it has translation factors) and the ionic concentrations used been known since the 1980s that translation can initiate (Kozak 1989, 1990b). at codons other than AUG, albeit at a much lower efficien- While endogenous functional non-AUG start codons cy (Zitomer et al. 1984; Peabody 1987, 1989; Clements et may be more prevalent than previously appreciated, it al. 1988; Hann et al. 1988). In most of these cases, near- should not be dismissed that such codons typically per- form at a markedly reduced efficiency compared with © 2017 Kearse and Wilusz This article is distributed exclusively by Cold [Keywords: start codon; near-cognate; RAN translation; translation Spring Harbor Laboratory Press for the first six months after the full-issue initiation; eIF2A; eIF2D] publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). Corresponding author: [email protected] After six months, it is available under a Creative Commons License (At- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.305250. tribution-NonCommercial 4.0 International), as described at http:// 117. creativecommons.org/licenses/by-nc/4.0/. GENES & DEVELOPMENT 31:1717–1731 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/17; www.genesdev.org 1717 Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Kearse and Wilusz Table 1. Efficiency of non-AUG start codons in various assays Rabbit Rabbit Saccharomyces reticulocyte reticulocyte Wheat germ cerevisiae HEK293T cells Biological lysate (Peabody lysate (Wei extract (Peabody (Clements et al. Neurospora crassa (Ivanov et al. source 1989) et al. 2013) 1989) 1988) (Wei et al. 2013) 2010) Reporter Dihydrofolate Firefly Dihydrofolate β-Galactosidase Firefly luciferase Firefly luciferase used reductase luciferase reductase AUG 100 100 100 100 100 100 CUG 82 18 36 0.22 10 19 GUG 36 11 8 0.5 6 9 UUG 39 8 10 0.37 2.5 2 ACG 84 5 45 0.39 3.5 7 AUC 47 3 17 0.05 <0.5 2 AUU 67 6 14 0.38 1 3 AAG 14 0.01 3 0.02 <0.5 <0.5 AUA 59 5 30 0.29 3 3 AGG 17 0.02 3 0.04 <0.5 <0.5 AUG codons (Table 1). Thus, the purpose of this review is across the transcriptome as well as revealed key features not to suggest that most proteins within cells are derived that influence translation patterns (for review, see Ingolia from non-AUG start codons but to highlight how alterna- 2014; Brar and Weissman 2015; Andreev et al. 2017). For tive initiation codons can be used to increase protein iso- example, specific combinations of codons (often involving form diversity and impact cellular processes (Touriol et al. proline) can slow translation in both mammalian cells and 2003). Besides highlighting the widespread nature of non- yeast (Ingolia et al. 2011; Gamble et al. 2016). AUG translation events in eukaryotic cells, we discuss By treating cells with early elongation inhibitors that the trans-acting proteins and the features within mRNAs block 80S ribosomes after initiation but before the first that dictate start codon recognition. In some cases, a ca- translocation cycle, ribosome profiling can be used to nonical scanning mechanism of translation initiation ap- define translation start sites (Fig. 1). Lactimidomycin pears to be used, but there are also a number of alternative (which binds the exit site [E site] of the 60S subunit) factors (which can be induced by various stresses) that al- (Schneider-Poetsch et al. 2010) or harringtonine (which ter start codon preferences. As aberrant non-AUG transla- binds the aminoacyl site [A site] of the 60S subunit) (Fres- tion events are associated with, and likely drive, multiple no et al. 1977) are used for exactly this purpose, as they human diseases, including cancer and neurodegeneration have little, if any, effect on 80S ribosomes beyond the first (Zu et al. 2011; Sendoel et al. 2017), there exists the in- triguing possibility that modulating non-AUG translation events (e.g., using small molecule inhibitors) may have profound therapeutic effects. Cycloheximide Harringtonine Thousands of non-AUG codons can be used for translation initiation For many years, the identification of non-AUG start co- dons was often fortuitous and resulted from efforts aimed at cloning genes of interest. For example, Xiao et al. (1991) observed that endogenous TEAD1 (also called TEF-1) in HeLa cells did not comigrate in SDS-PAGE with in vitro translated TEAD1 that was initiated from the predicted Footprints AUG start codon. By pursuing this observation and using 80S Ribosome start stop start stop mutational analysis, it was revealed that endogenous TEAD1 uses solely an upstream AUU start codon. Ap- proaches like these are largely limited to single genes. Figure 1. Ribosome profiling can be used to reveal translation However, a genome-wide view of translation can now be initiation sites across the transcriptome. (Left) Treatment with cycloheximide (blue), which binds to the E site of the 60S subunit, provided by ribosome profiling, in which short mRNA pauses all elongating 80S ribosomes. The mapped ribosome foot- fragments that are protected by 80S ribosomes are purified prints thus typically cover the entire ORF. (Right) In contrast, har- and subjected to high-throughput sequencing (Ingolia ringtonine (red) binds to the A site of the 60S subunit and only et al. 2009, 2011; Ingolia 2010). Ribosome profiling, some- inhibits 80S ribosomes just after subunit joining at the start co- times referred to as Ribo-seq, has revolutionized our un- don. Mapped ribosome footprints from harringtonine treatment derstanding of where translating ribosomes are present are thus enriched for the start codon. 1718 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Regulated use of non-AUG start codons translocation step. Thousands of previously unannotated initiation events have now been identified in mouse em- eIF2 eIF5 GTP bryonic stem cells, ∼60% of which initiate at a non- Met 1 1A 43S AUG start codon (Ingolia et al.
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