Abstract
Global methods for assaying translation have greatly improved our understanding of the protein-coding capacity of the genome. In particular, it is now possible to perform genome-wide and condition-specific identification of translation initiation sites through modified ribosome profiling methods that selectively capture initiating ribosomes. Here we discuss our recent study applying such an approach to meiotic and mitotic timepoints in the simple eukaryote, budding yeast, as an example of the surprising diversity of protein products—many of which are non-canonical—that can be revealed by such methods. We also highlight several key challenges in studying non-canonical protein isoforms that have precluded their prior systematic discovery. A growing body of work supports expanded use of empirical protein-coding region identification, which can help relieve some of the limitations and biases inherent to traditional genome annotation approaches. Our study also argues for the adoption of less static views of gene identity and a broader framework for considering the translational capacity of the genome.
Similar content being viewed by others
References
Aitken CE, Lorsch JR (2012) A mechanistic overview of translation initiation in eukaryotes. Nat Struct Mol Biol 19:568–576. https://doi.org/10.1038/nsmb.2303
Baralle FE, Giudice J (2017) Alternative splicing as a regulator of development and tissue identity. Nat Rev Mol Cell Biol 18:437–451. https://doi.org/10.1038/nrm.2017.27
Brar GA, Weissman JS (2015) Ribosome profiling reveals the what, when, where and how of protein synthesis. Nat Rev Mol Cell Biol 16:651–664. https://doi.org/10.1038/nrm4069
Brar GA, Yassour M, Friedman N, Regev A, Ingolia NT, Weissman JS (2012) High-resolution view of the yeast meiotic program revealed by ribosome profiling. Science 335:552–557. https://doi.org/10.1126/science.1215110
Brockdorff N, McCabe M, Norris P, Cooper J, Swift S, Kay F (1992) The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus. Cell 71:515–526
Celik A, Baker R, He F, Jacobson A (2017) High-resolution profiling of NMD targets in yeast reveals translational fidelity as a basis for substrate selection. RNA 23:735–748. https://doi.org/10.1261/rna.060541.116
Chang K-J, Wang C-C (2004) Translation initiation from a naturally occurring non-AUG codon in Saccharomyces cerevisiae. J Biol Chem 279:13778–13785. https://doi.org/10.1074/jbc.M311269200
Chen S-J, Lin G, Chang K-J, Yeh L-S, Wang C-C (2008) Translational efficiency of a non-AUG initiation codon is significantly affected by its sequence context in yeast. J Biol Chem 283:3173–3180. https://doi.org/10.1074/jbc.M706968200
Chen J, Tresenrider A, Chia M, McSwiggen DT, Spedale G, Jorgensen V, Liao H, van Werven FJ, Ünal E (2017) Kinetochore inactivation by expression of a repressive mRNA. eLife 6:e27417. https://doi.org/10.7554/eLife.27417
Cheng Z, Otto GM, Powers EN, Keskin A, Mertins P, Carr SA, Jovanovic M, Brar GA (2018) Pervasive, coordinated protein-level changes driven by transcript isoform switching during meiosis. Cell 172:910-923.e16. https://doi.org/10.1016/j.cell.2018.01.035
Chia M, Tresenrider A, Chen J, Spedale G, Jorgensen V, Ünal E, van Werven FJ (2017) Transcription of a 5’ extended mRNA isoform directs dynamic chromatin changes and interference of a downstream promoter. eLife 6:e27420. https://doi.org/10.7554/eLife.27420
Clements JM, Laz TM, Sherman F (1988) Efficiency of translation initiation by non-AUG codons in Saccharomyces cerevisiae. Mol Cell Biol 8:4533–4536. https://doi.org/10.1128/MCB.8.10.4533
Dinger ME, Pang KC, Mercer TR, Mattick JS (2008) Differentiating protein-coding and noncoding RNA: challenges and ambiguities. PLoS Comput Biol 4:e1000176. https://doi.org/10.1371/journal.pcbi.1000176
Eisenberg AR, Higdon AL, Hollerer I, Fields AP, Jungreis I, Diamond PD, Kellis M, Jovanovic M, Brar GA (2020) Translation initiation site profiling reveals widespread synthesis of non-AUG-initiated protein isoforms in yeast. Cell Syst. https://doi.org/10.1016/j.cels.2020.06.011
Engel SR, Dietrich FS, Fisk DG, Binkley G, Balakrishnan R, Costanzo MC, Dwight SS, Hitz BC, Karra K, Nash RS, Weng S, Wong ED, Lloyd P, Skrzypek MS, Miyasato SR, Simison M, Cherry JM (2014) The reference genome sequence of Saccharomyces cerevisiae : then and now. G3 4:389–398. https://doi.org/10.1534/g3.113.008995
Fields AP, Rodriguez EH, Jovanovic M, Stern-Ginossar N, Haas BJ, Mertins P, Raychowdhury R, Hacohen N, Carr SA, Ingolia NT, Regev A, Weissman JS (2015) A regression-based analysis of ribosome-profiling data reveals a conserved complexity to mammalian translation. Mol Cell 60:816–827. https://doi.org/10.1016/j.molcel.2015.11.013
Fritsch C, Herrmann A, Nothnagel M, Szafranski K, Huse K, Schumann F, Schreiber S, Platzer M, Krawczak M, Hampe J, Brosch M (2012) Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting. Genome Res 22:2208–2218. https://doi.org/10.1101/gr.139568.112
Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG (1996) Life with 6000 genes. Science 274:546–567. https://doi.org/10.1126/science.274.5287.546
Guenther U-P, Weinberg DE, Zubradt MM, Tedeschi FA, Stawicki BN, Zagore LL, Brar GA, Licatalosi DD, Bartel DP, Weissman JS, Jankowsky E (2018) The helicase Ded1p controls use of near-cognate translation initiation codons in 5′ UTRs. Nature 559:130–134. https://doi.org/10.1038/s41586-018-0258-0
Heublein M, Ndi M, Vazquez-Calvo C, Vögtle F-N, Ott M (2019) Alternative translation initiation at a UUG codon gives rise to two functional variants of the mitochondrial protein Kgd4. J Mol Biol 431:1460–1467. https://doi.org/10.1016/j.jmb.2019.02.023
Hug N, Longman D, Cáceres JF (2016) Mechanism and regulation of the nonsense-mediated decay pathway. Nucleic Acids Res 44:1483–1495. https://doi.org/10.1093/nar/gkw010
Ingolia NT (2014) Ribosome profiling: new views of translation, from single codons to genome scale. Nat Rev Genet 15:205–213. https://doi.org/10.1038/nrg3645
Ingolia NT, Ghaemmaghami S, Newman JRS, Weissman JS (2009) Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324:218–223. https://doi.org/10.1126/science.1168978
Ingolia NT, Lareau LF, Weissman JS (2011) Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147:789–802. https://doi.org/10.1016/j.cell.2011.10.002
Ivanov IP, Firth AE, Michel AM, Atkins JF, Baranov PV (2011) Identification of evolutionarily conserved non-AUG-initiated N-terminal extensions in human coding sequences. Nucleic Acids Res 39:4220–4234. https://doi.org/10.1093/nar/gkr007
Juneau K, Palm C, Miranda M, Davis RW (2007) High-density yeast-tiling array reveals previously undiscovered introns and extensive regulation of meiotic splicing. Proc Natl Acad Sci 104:1522–1527. https://doi.org/10.1073/pnas.0610354104
Kaiser CA, Botstein D (1990) Efficiency and diversity of protein localization by random signal sequences. Mol Cell Biol 10:3163–3173. https://doi.org/10.1128/MCB.10.6.3163
Kearse MG, Wilusz JE (2017) Non-AUG translation: a new start for protein synthesis in eukaryotes. Genes Dev 31:1717–1731. https://doi.org/10.1101/gad.305250.117
Kearse MG, Goldman DH, Choi J, Nwaezeapu C, Liang D, Green KM, Goldstrohm AC, Todd PK, Green R, Wilusz JE (2019) Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors. Genes Dev 33:871–885. https://doi.org/10.1101/gad.324715.119
Kolitz SE, Takacs JE, Lorsch JR (2008) Kinetic and thermodynamic analysis of the role of start codon/anticodon base pairing during eukaryotic translation initiation. RNA 15:138–152. https://doi.org/10.1261/rna.1318509
Kozak M (1990) Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes. Proc Natl Acad Sci 87:8301–8305. https://doi.org/10.1073/pnas.87.21.8301
Kritsiligkou P, Chatzi A, Charalampous G, Mironov A, Grant CM, Tokatlidis K (2017) Unconventional targeting of a thiol peroxidase to the mitochondrial intermembrane space facilitates oxidative protein folding. Cell Rep 18:2729–2741. https://doi.org/10.1016/j.celrep.2017.02.053
Lee S, Liu B, Lee S, Huang S-X, Shen B, Qian S-B (2012) Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution. Proc Natl Acad Sci 109:E2424–E2432. https://doi.org/10.1073/pnas.1207846109
Liti G (2015) The fascinating and secret wild life of the budding yeast S. cerevisiae. eLife 4:e05835. https://doi.org/10.7554/eLife.05835
Marston AL, Amon A (2004) Meiosis: cell-cycle controls shuffle and deal. Nat Rev Mol Cell Biol 5:983–997. https://doi.org/10.1038/nrm1526
Monteuuis G, Miścicka A, Świrski M, Zenad L, Niemitalo O, Wrobel L, Alam J, Chacinska A, Kastaniotis AJ, Kufel J (2019) Non-canonical translation initiation in yeast generates a cryptic pool of mitochondrial proteins. Nucleic Acids Res 47:5777–5791. https://doi.org/10.1093/nar/gkz301
Morris DR, Geballe AP (2000) Upstream open reading frames as regulators of mRNA translation. Mol Cell Biol 20:8635–8642. https://doi.org/10.1128/MCB.20.23.8635-8642.2000
Nagalakshmi U, Wang Z, Waern K, Shou C, Raha D, Gerstein M, Snyder M (2008) The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320:1344–1349. https://doi.org/10.1126/science.1158441
Otto GM, Brar GA (2018) Seq-ing answers: uncovering the unexpected in global gene regulation. Curr Genet 64:1183–1188. https://doi.org/10.1007/s00294-018-0839-3
Pleiss JA, Whitworth GB, Bergkessel M, Guthrie C (2007) Transcript specificity in yeast pre-mRNA splicing revealed by mutations in core spliceosomal components. PLoS Biol 5:e90. https://doi.org/10.1371/journal.pbio.0050090
Renz PF, Valdivia-Francia F, Sendoel A (2020) Some like it translated: small ORFs in the 5′UTR. Exp Cell Res 396:112229. https://doi.org/10.1016/j.yexcr.2020.112229
Sapkota D, Lake AM, Yang W, Yang C, Wesseling H, Guise A, Uncu C, Dalal JS, Kraft AW, Lee J-M, Sands MS, Steen JA, Dougherty JD (2019) Cell-type-specific profiling of alternative translation identifies regulated protein isoform variation in the mouse brain. Cell Rep 26:594-607.e7. https://doi.org/10.1016/j.celrep.2018.12.077
Schmitt AM, Chang HY (2017) Long noncoding RNAs: at the intersection of cancer and chromatin biology. Cold Spring Harb Perspect Med 7:a026492. https://doi.org/10.1101/cshperspect.a026492
Schneider-Poetsch T, Ju J, Eyler DE, Dang Y, Bhat S, Merrick WC, Green R, Shen B, Liu JO (2010) Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat Chem Biol 6:209–217. https://doi.org/10.1038/nchembio.304
Stern-Ginossar N, Weisburd B, Michalski A, Le VTK, Hein MY, Huang S-X, Ma M, Shen B, Qian S-B, Hengel H, Mann M, Ingolia NT, Weissman JS (2012) Decoding human cytomegalovirus. Science 338:1088–1093. https://doi.org/10.1126/science.1227919
Suomi F, Menger KE, Monteuuis G, Naumann U, Kursu VAS, Shvetsova A, Kastaniotis AJ (2014) Expression and evolution of the non-canonically translated yeast mitochondrial acetyl-CoA carboxylase Hfa1p. PLoS ONE 9:e114738. https://doi.org/10.1371/journal.pone.0114738
Suzuki K, Hashimoto T, Otaka E (1990) Yeast ribosomal proteins: XI. Molecular analysis of two genes encoding YL41, an extremely small and basic ribosomal protein, from Saccharomyces cerevisiae. Curr Genet 17:185–190
Tang H-L, Yeh L-S, Chen N-K, Ripmaster T, Schimmel P, Wang C-C (2004) Translation of a yeast mitochondrial tRNA synthetase initiated at redundant non-AUG codons. J Biol Chem 279:49656–49663. https://doi.org/10.1074/jbc.M408081200
Touriol C, Bornes S, Bonnal S, Audigier S, Prats H, Prats A-C, Vagner S (2003) Generation of protein isoform diversity by alternative initiation of translation at non-AUG codons. Biol Cell 95:169–178. https://doi.org/10.1016/S0248-4900(03)00033-9
Tresenrider A, Ünal E (2018) One-two punch mechanism of gene repression: a fresh perspective on gene regulation. Curr Genet 64:581–588. https://doi.org/10.1007/s00294-017-0793-5
van Werven FJ, Amon A (2011) Regulation of entry into gametogenesis. Phil Trans R Soc B 366:3521–3531. https://doi.org/10.1098/rstb.2011.0081
Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63. https://doi.org/10.1038/nrg2484
Wood V, Lock A, Harris MA, Rutherford K, Bahler J, Oliver SG (2019) Hidden in plain sight: what remains to be discovered in the eukaryotic proteome? Open Biol 9:180241
Yu X, Warner JR (2001) Expression of a micro-protein. J Biol Chem 276:33821–33825. https://doi.org/10.1074/jbc.M103772200
Zhang H, Wang Y, Lu J (2019) Function and evolution of upstream ORFs in eukaryotes. Trends Biochem Sci 44:782–794. https://doi.org/10.1016/j.tibs.2019.03.002
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by M.Kupiec.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Higdon, A.L., Brar, G.A. Rules are made to be broken: a “simple” model organism reveals the complexity of gene regulation. Curr Genet 67, 49–56 (2021). https://doi.org/10.1007/s00294-020-01121-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-020-01121-8