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Quantitative Comparisons of Translation Activity by Ribosome Profiling with Internal Standards

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Ribosome Profiling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2252))

Abstract

Ribosome profiling is a genome-wide approach to map the positions of ribosomes on messenger RNAs. The abundance of ribosome-protected fragments can be used within condition to compare relative translation activities between different transcripts and between distinct conditions for the same transcript. A unified and routine method is currently lacking, however, to normalize between conditions for differences in global translation levels. Here we describe experimental and computational methods to use an orthogonal species spike-in, or internal standard, to enable absolute comparisons of translation activity between conditions. This simple modification of standard ribosome profiling provides a robust approach for accurately interpreting the effects of diverse genetic, chemical, and environmental perturbations of translation.

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References

  1. Arribere JA, Doudna JA, Gilbert WV (2011) Reconsidering movement of eukaryotic mRNAs between polysomes and P bodies. Mol Cell 44(5):745–758. https://doi.org/10.1016/j.molcel.2011.09.019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Vaidyanathan PP, Zinshteyn B, Thompson MK, Gilbert WV (2014) Protein kinase A regulates gene-specific translational adaptation in differentiating yeast. RNA 20(6):912–922. https://doi.org/10.1261/rna.044552.114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Zhang Y, Burkhardt DH, Rouskin S, Li GW, Weissman JS, Gross CA (2018) A stress response that monitors and regulates mRNA structure is central to cold shock adaptation. Mol Cell 70(2):274–286 e277. https://doi.org/10.1016/j.molcel.2018.02.035

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, Tempst P, Sabatini DM (2002) mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110(2):163–175. https://doi.org/10.1016/s0092-8674(02)00808-5

    Article  CAS  PubMed  Google Scholar 

  5. Thoreen CC, Chantranupong L, Keys HR, Wang T, Gray NS, Sabatini DM (2012) A unifying model for mTORC1-mediated regulation of mRNA translation. Nature 485(7396):109–113. https://doi.org/10.1038/nature11083

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dever TE, Feng L, Wek RC, Cigan AM, Donahue TF, Hinnebusch AG (1992) Phosphorylation of initiation factor 2 alpha by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast. Cell 68(3):585–596. https://doi.org/10.1016/0092-8674(92)90193-g

    Article  CAS  PubMed  Google Scholar 

  7. Hinnebusch AG, Ivanov IP, Sonenberg N (2016) Translational control by 5′-untranslated regions of eukaryotic mRNAs. Science 352(6292):1413–1416. https://doi.org/10.1126/science.aad9868

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bhat M, Robichaud N, Hulea L, Sonenberg N, Pelletier J, Topisirovic I (2015) Targeting the translation machinery in cancer. Nat Rev Drug Discov 14(4):261–278. https://doi.org/10.1038/nrd4505

    Article  CAS  PubMed  Google Scholar 

  9. Pospisek M, Valasek L (2013) Polysome profile analysis--yeast. Methods Enzymol 530:173–181. https://doi.org/10.1016/B978-0-12-420037-1.00009-9

    Article  CAS  PubMed  Google Scholar 

  10. Lodish HF (1971) Alpha and beta globin messenger ribonucleic acid. Different amounts and rates of initiation of translation. J Biol Chem 246(23):7131–7138

    Article  CAS  PubMed  Google Scholar 

  11. Khoutorsky A, Bonin RP, Sorge RE, Gkogkas CG, Pawlowski SA, Jafarnejad SM, Pitcher MH, Alain T, Perez-Sanchez J, Salter EW, Martin L, Ribeiro-da-Silva A, De Koninck Y, Cervero F, Mogil JS, Sonenberg N (2015) Translational control of nociception via 4E-binding protein 1. elife 4:e12002. https://doi.org/10.7554/eLife.12002

    Article  PubMed  PubMed Central  Google Scholar 

  12. Iwasaki S, Ingolia NT (2017) The growing toolbox for protein synthesis studies. Trends Biochem Sci 42(8):612–624. https://doi.org/10.1016/j.tibs.2017.05.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Park E-H, Zhang F, Warringer J, Sunnerhagen P, Hinnebusch AG (2011) Depletion of eIF4G from yeast cells narrows the range of translational efficiencies genome-wide. BMC Genomics 12(1):1–8. https://doi.org/10.1186/1471-2164-12-68

    Article  CAS  Google Scholar 

  14. Rubio CA, Weisburd B, Holderfield M, Arias C, Fang E, DeRisi JL, Fanidi A (2014) Transcriptome-wide characterization of the eIF4A signature highlights plasticity in translation regulation. Genome Biol 15(10):476. https://doi.org/10.1186/s13059-014-0476-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ingolia NT, Ghaemmaghami S, Newman JRS, Weissman JS (2009) Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324(5924):218–223. https://doi.org/10.1126/science.1168978

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Li GW, Burkhardt D, Gross C, Weissman JS (2014) Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell 157(3):624–635. https://doi.org/10.1016/j.cell.2014.02.033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Weinberg DE, Shah P, Eichhorn SW, Hussmann JA, Plotkin JB, Bartel DP (2016) Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation. Cell Rep 14(7):1787–1799. https://doi.org/10.1016/j.celrep.2016.01.043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zinshteyn B, Rojas-Duran MF, Gilbert WV (2017) Translation initiation factor eIF4G1 preferentially binds yeast transcript leaders containing conserved oligo-uridine motifs. RNA 23(9):1365–1375. https://doi.org/10.1261/rna.062059.117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sen ND, Zhou F, Ingolia NT, Hinnebusch AG (2015) Genome-wide analysis of translational efficiency reveals distinct but overlapping functions of yeast DEAD-box RNA helicases Ded1 and eIF4A. Genome Res 25(8):1196–1205. https://doi.org/10.1101/gr.191601.115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. McGlincy NJ, Ingolia NT (2017) Transcriptome-wide measurement of translation by ribosome profiling. Methods 126:112–129. https://doi.org/10.1016/j.ymeth.2017.05.028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Han Y, David A, Liu B, Magadan JG, Bennink JR, Yewdell JW, Qian SB (2012) Monitoring cotranslational protein folding in mammalian cells at codon resolution. Proc Natl Acad Sci U S A 109(31):12467–12472. https://doi.org/10.1073/pnas.1208138109

    Article  PubMed  PubMed Central  Google Scholar 

  22. Popa A, Lebrigand K, Barbry P, Waldmann R (2016) Pateamine A-sensitive ribosome profiling reveals the scope of translation in mouse embryonic stem cells. BMC Genomics 17(1):52. https://doi.org/10.1186/s12864-016-2384-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Iwasaki S, Floor SN, Ingolia NT (2016) Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor. Nature 534(7608):558–561. https://doi.org/10.1038/nature17978

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Thompson MK, Rojas-Duran MF, Gangaramani P, Gilbert WV (2016) The ribosomal protein Asc1/RACK1 is required for efficient translation of short mRNAs. eLife 5:e11154. https://doi.org/10.7554/eLife.11154

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wang YJ, Vaidyanathan PP, Rojas-Duran MF, Udeshi ND, Bartoli KM, Carr SA, Gilbert WV (2018) Lso2 is a conserved ribosome-bound protein required for translational recovery in yeast. PLoS Biol 16(9):e2005903. https://doi.org/10.1371/journal.pbio.2005903

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Illumina (2019) Illumina Adapter Sequences. https://support.illumina.com/downloads/illumina-adapter-sequences-document-1000000002694.html

    Google Scholar 

  27. Wu CC, Zinshteyn B, Wehner KA, Green R (2019) High-resolution ribosome profiling defines discrete ribosome elongation states and translational regulation during cellular stress. Mol Cell 73(5):959–970 e955. https://doi.org/10.1016/j.molcel.2018.12.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Schuller AP, Wu CC-C, Dever TE, Buskirk AR, Green R (2017) eIF5A functions globally in translation elongation and termination. Mol Cell 66(2):194–205. https://doi.org/10.1016/j.molcel.2017.03.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Zinshteyn B, Gilbert WV (2013) Loss of a conserved tRNA anticodon modification perturbs cellular signaling. PLoS Genet 9(8):e1003675. https://doi.org/10.1371/journal.pgen.1003675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12):550. https://doi.org/10.1186/s13059-014-0550-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Xiao Z, Zou Q, Liu Y, Yang X (2016) Genome-wide assessment of differential translations with ribosome profiling data. Nat Commun 7:11194. https://doi.org/10.1038/ncomms11194

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Athanasiadou R, Neymotin B, Brandt N, Wang W, Christiaen L, Gresham D, Tranchina D (2019) A complete statistical model for calibration of RNA-seq counts using external spike-ins and maximum likelihood theory. PLoS Comput Biol 15(3):e1006794. https://doi.org/10.1371/journal.pcbi.1006794

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Herbert ZT, Kershner JP, Butty VL, Thimmapuram J, Choudhari S, Alekseyev YO, Fan J, Podnar JW, Wilcox E, Gipson J, Gillaspy A, Jepsen K, BonDurant SS, Morris K, Berkeley M, LeClerc A, Simpson SD, Sommerville G, Grimmett L, Adams M, Levine SS (2018) Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction. BMC Genomics 19(1):199. https://doi.org/10.1186/s12864-018-4585-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gerashchenko MV, Gladyshev VN (2014) Translation inhibitors cause abnormalities in ribosome profiling experiments. Nucleic Acids Res 42(17):e134–e134. https://doi.org/10.1093/nar/gku671

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Guydosh NR, Green R (2014) Dom34 rescues ribosomes in 3′ untranslated regions. Cell 156(5):950–962. https://doi.org/10.1016/j.cell.2014.02.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Guydosh NR, Green R (2017) Translation of poly(A) tails leads to precise mRNA cleavage. RNA 23(5):749–761. https://doi.org/10.1261/rna.060418.116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294(5543):858–862. https://doi.org/10.1126/science.1065062

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Drs. Rachel Green, Colin Wu, Boris Zinshteyn, and Mary Thompson for helpful discussions, Maria Rojas-Duran for assistance with library preparation, and Drs. Joshua Arribere and Boris Zinshteyn for critical reading of the manuscript. This work was funded by NIH R01GM094303 and R01GM132358 to WVG and an NSF Graduate Research Fellowship to YJW.

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Author notes

  1. Yinuo J. Wang

    Present address: Arrakis Therapeutics, Waltham, MA, USA

Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Yinuo J. Wang & Wendy V. Gilbert

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  1. Yinuo J. Wang
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  2. Wendy V. Gilbert
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Corresponding author

Correspondence to Wendy V. Gilbert .

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Editors and Affiliations

  1. Department of Dermatology, Boston University School of Medicine, Boston, MA, USA

    Vyacheslav M. Labunskyy

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Wang, Y.J., Gilbert, W.V. (2021). Quantitative Comparisons of Translation Activity by Ribosome Profiling with Internal Standards. In: Labunskyy, V.M. (eds) Ribosome Profiling. Methods in Molecular Biology, vol 2252. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1150-0_5

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  • DOI: https://doi.org/10.1007/978-1-0716-1150-0_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1149-4

  • Online ISBN: 978-1-0716-1150-0

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