Abstract
Translational regulation is important for plant growth, metabolism, and acclimation to environmental challenges. Ribosome profiling involves the nuclease digestion of mRNAs associated with ribosomes and mapping of the generated ribosome-protected footprints to transcripts. This is useful for investigation of translational regulation. Here we present a detailed method to generate, purify, and high-throughput-sequence ribosome footprints from Arabidopsis thaliana using two different isolation methods, namely, conventional differential centrifugation and the translating ribosome affinity purification (TRAP) technology. These methodologies provide researchers with an opportunity to quantitatively assess with high-resolution the translational activity of individual mRNAs by determination of the position and number of ribosomes in the corresponding mRNA. The results can provide insights into the translation of upstream open reading frames, alternatively spliced transcripts, short open reading frames, and other aspects of translation.
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References
Bailey-Serres J (2013) Microgenomics: genome-scale, cell-specific monitoring of multiple gene regulation tiers. Annu Rev Plant Biol 64:293–325
Gygi SP, Rochon Y, Franza BR et al (1999) Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 19:1720–1730
Piques M, Schulze WX, Hohne M et al (2009) Ribosome and transcript copy numbers, polysome occupancy and enzyme dynamics in Arabidopsis. Mol Syst Biol 5:314. doi:10.1038/mbs.2009.1068
Bailey-Serres J, Sorenson R, Juntawong P (2009) Getting the message across: cytoplasmic ribonucleoprotein complexes. Trends Plant Sci 14:443–453
Kawaguchi R, Bailey-Serres J (2002) Regulation of translational initiation in plants. Curr Opin Plant Biol 5:460–465
Hummel M, Rahmani F, Smeekens S et al (2009) Sucrose-mediated translational control. Ann Bot 104:1–7
Roy B, von Arnim AG (2013) Translational Regulation of Cytoplasmic mRNAs. Arabidopsis Book 11:e0165
Horiguchi G, Van Lijsebettens M, Candela H et al (2012) Ribosomes and translation in plant developmental control. Plant Sci 191–192:24–34
Zanetti ME, Chang IF, Gong FC et al (2005) Immunopurification of polyribosomal complexes of arabidopsis for global analysis of gene expression. Plant Physiol 138:624–635
Mustroph A, Juntawong P, Bailey-Serres J (2009) Isolation of plant polysomal mRNA by differential centrifugation and ribosome immunopurification methods. Methods Mol Biol 553:109–126
Kim BH, Cai X, Vaughn JN et al (2007) On the functions of the h subunit of eukaryotic initiation factor 3 in late stages of translation initiation. Genome Biol 8:R60
Tiruneh BS, Kim BH, Gallie DR et al (2013) The global translation profile in a ribosomal protein mutant resembles that of an eIF3 mutant. BMC Biol 11:123
Branco-Price C, Kawaguchi R, Ferreira RB et al (2005) Genome-wide analysis of transcript abundance and translation in Arabidopsis seedlings subjected to oxygen deprivation. Ann Bot 96:647–660
Branco-Price C, Kaiser KA, Jang CJH et al (2008) Selective mRNA translation coordinates energetic and metabolic adjustments to cellular oxygen deprivation and reoxygenation in Arabidopsis thaliana. Plant J 56:743–755
Kawaguchi R, Girke T, Bray EA et al (2004) Differential mRNA translation contributes to gene regulation under non-stress and dehydration stress conditions in Arabidopsis thaliana. Plant J 38:823–839
Pal SK, Liput M, Piques M et al (2013) Diurnal changes of polysome loading track sucrose content in the rosette of wild-type arabidopsis and the starchless pgm mutant. Plant Physiol 162:1246–1265
Liu MJ, Wu SH, Chen HM (2012) Widespread translational control contributes to the regulation of Arabidopsis photomorphogenesis. Mol Syst Biol 8:566
Sorenson R, Bailey-Serres J (2014) Selective mRNA sequestration by OLIGOURIDYLATE-BINDING PROTEIN 1 contributes to translational control during hypoxia in Arabidopsis. Proc Natl Acad Sci U S A 111:2373–2378
Goeres DC, Van Norman JM, Zhang W et al (2007) Components of the Arabidopsis mRNA decapping complex are required for early seedling development. Plant Cell 19:1549–1564
Zhang W, Murphy C, Sieburth LE (2010) Conserved RNaseII domain protein functions in cytoplasmic mRNA decay and suppresses Arabidopsis decapping mutant phenotypes. Proc Natl Acad Sci U S A 107:15981–15985
Belostotsky DA, Meagher RB (1996) A pollen-, ovule-, and early embryo-specific poly(A) binding protein from Arabidopsis complements essential functions in yeast. Plant Cell 8:1261–1275
Iwasaki S, Takeda A, Motose H et al (2007) Characterization of Arabidopsis decapping proteins AtDCP1 and AtDCP2, which are essential for post-embryonic development. FEBS Lett 581:2455–2459
Steffens A, Jaegle B, Tresch A et al (2014) Processing-body movement in Arabidopsis depends on an interaction between myosins and DECAPPING PROTEIN1. Plant Physiol 16:1879–1892
Xu J, Yang JY, Niu QW et al (2006) Arabidopsis DCP2, DCP1, and VARICOSE form a decapping complex required for postembryonic development. Plant Cell 18:3386–3398
Xu J, Chua NH (2009) Arabidopsis decapping 5 is required for mRNA decapping, P-body formation, and translational repression during postembryonic development. Plant Cell 21:3270–3279
Kawaguchi R, Bailey-Serres J (2005) mRNA sequence features that contribute to translational regulation in Arabidopsis. Nucleic Acids Res 33:955–965
Mustroph A, Lee SC, Oosumi T et al (2010) Cross-kingdom comparison of transcriptomic adjustments to low-oxygen stress highlights conserved and plant-specific responses. Plant Physiol 152:1484–1500
Matsuura H, Ishibashi Y, Shinmyo A et al (2010) Genome-wide analyses of early translational responses to elevated temperature and high salinity in Arabidopsis thaliana. Plant Cell Physiol 51:448–462
Yanguez E, Castro-Sanz AB, Fernandez-Bautista N et al (2013) Analysis of genome-wide changes in the translatome of Arabidopsis seedlings subjected to heat stress. PLoS One 8:e71425
Juntawong P, Sorenson R, Bailey-Serres J (2013) Cold shock protein 1 chaperones mRNAs during translation in Arabidopsis thaliana. Plant J 74:1016–1028
Sormani R, Delannoy E, Lageix S et al (2011) Sublethal cadmium intoxication in Arabidopsis thaliana impacts translation at multiple levels. Plant Cell Physiol 52:436–447
Juntawong P, Bailey-Serres J (2012) Dynamic light regulation of translation status in Arabidopsis thaliana. Front Plant Sci 3:66
Liu MJ, Wu SH, Wu JF et al (2013) Translational landscape of photomorphogenic Arabidopsis. Plant Cell 25:3699–3710
Nicolai M, Roncato MA, Canoy AS et al (2006) Large-scale analysis of mRNA translation states during sucrose starvation in Arabidopsis cells identifies cell proliferation and chromatin structure as targets of translational control. Plant Physiol 141:663–673
Rosado A, Li R, van de Ven W et al (2012) Arabidopsis ribosomal proteins control developmental programs through translational regulation of auxin response factors. Proc Natl Acad Sci U S A 109:19537–19544
Schepetilnikov M, Dimitrova M, Mancera-Martinez E et al (2013) TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h. EMBO J 32:1087–1102
Juntawong P, Girke T, Bazin J et al (2014) Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis. Proc Natl Acad Sci U S A 111:E203–E212
Ribeiro DM, Araujo WL, Fernie AR et al (2012) Translatome and metabolome effects triggered by gibberellins during rosette growth in Arabidopsis. J Exp Bot 63:2769–2786
Moghe GD, Lehti-Shiu MD, Seddon AE et al (2013) Characteristics and significance of intergenic polyadenylated RNA transcription in Arabidopsis. Plant Physiol 161:210–224
Mustroph A, Zanetti ME, Jang CJ et al (2009) Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis. Proc Natl Acad Sci U S A 106:18843–18848
Mustroph A, Barding GA Jr, Kaiser KA et al (2014) Characterization of distinct root and shoot responses to low-oxygen stress in Arabidopsis with a focus on primary C- and N-metabolism. Plant Cell Environ 37:2366–2380
Aubry S, Smith-Unna RD, Boursnell CM et al (2014) Transcript residency on ribosomes reveals a key role for the Arabidopsis thaliana bundle sheath in sulfur and glucosinolate metabolism. Plant J 78:659–673
Jiao Y, Meyerowitz EM (2010) Cell-type specific analysis of translating RNAs in developing flowers reveals new levels of control. Mol Syst Biol 6:419
Lin SY, Chen PW, Chuang MH et al (2014) Profiling of translatomes of in vivo-grown pollen tubes reveals genes with roles in micropylar guidance during pollination in Arabidopsis. Plant Cell 26:602–618
Hummel M, Cordewener JH, de Groot JC et al (2012) Dynamic protein composition of Arabidopsis thaliana cytosolic ribosomes in response to sucrose feeding as revealed by label free MSE proteomics. Proteomics 12:1024–1038
Park SH, Chung PJ, Juntawong P et al (2012) Posttranscriptional control of photosynthetic mRNA decay under stress conditions requires 3′ and 5′ untranslated regions and correlates with differential polysome association in rice. Plant Physiol 159:1111–1124
Khandal D, Samol I, Buhr F et al (2009) Singlet oxygen-dependent translational control in the tigrina-d.12 mutant of barley. Proc Natl Acad Sci U S A 106:13112–13117
Reynoso MA, Blanco FA, Bailey-Serres J et al (2012) Selective recruitment of mRNAs and miRNAs to polyribosomes in response to rhizobia infection in Medicago truncatula. Plant J 73:289–301
Ron M, Kajala K, Pauluzzi G et al (2014) Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiol 166:455–469
Wolin SL, Walter P (1988) Ribosome pausing and stacking during translation of a eukaryotic mRNA. EMBO J 7:3559–3569
Arava Y, Wang Y, Storey JD et al (2003) Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 100:3889–3894
Arava Y, Boas FE, Brown PO et al (2005) Dissecting eukaryotic translation and its control by ribosome density mapping. Nucleic Acids Res 33:2421–2432
Rogers K, Chen X (2013) Biogenesis, turnover, and mode of action of plant microRNAs. Plant Cell 25:2383–2399
Reddy AS, Marquez Y, Kalyna M et al (2013) Complexity of the alternative splicing landscape in plants. Plant Cell 25:3657–3683
Ingolia NT, Ghaemmaghami S, Newman JR et al (2009) Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324:218–223
Bentley DR, Balasubramanian S, Swerdlow HP et al (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456:53–59
Guo H, Ingolia NT, Weissman JS et al (2010) Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466:835–840
Hsieh AC, Liu Y, Edlind MP et al (2012) The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature 485:55–56
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
Bazzini AA, Lee MT, Giraldez AJ (2012) Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish. Science 336:233–237
Ingolia NT (2010) Genome-wide translational profiling by ribosome footprinting. Methods Enzymol 470:119–142
Zoschke R, Watkins KP, Barkan A (2013) A rapid ribosome profiling method elucidates chloroplast ribosome behavior in vivo. Plant Cell 25:2265–2275
Kim YJ, Maizel A, Chen X (2014) Traffic into silence: endomembranes and post-transcriptional RNA silencing. EMBO J 33:968–980
Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M et al (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320:1185–1190
Dugas DV, Bartel B (2008) Sucrose induction of Arabidopsis miR398 represses two Cu/Zn superoxide dismutases. Plant Mol Biol 67:403–417
Li S, Liu L, Zhuang X et al (2013) MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell 153:562–574
Chen X (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303:2022–2025
Aukerman MJ, Sakai H (2003) Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes. Plant Cell 15:2730–2741
Guttman M, Russell P, Ingolia NT et al (2013) Ribosome profiling provides evidence that large noncoding RNAs do not encode proteins. Cell 154:240–251
Mustroph A, Zanetti ME, Girke T et al (2013) Isolation and analysis of mRNAs from specific cell types of plants by ribosome immunopurification. Methods Mol Biol 959:277–302
Ingolia NT, Brar GA, Rouskin S et al (2012) The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc 7:1534–1550
Ingolia NT, Brar GA, Rouskin S et al (2013) Genome-wide annotation and quantitation of translation by ribosome profiling. Curr Protoc Mol Biol Chapter 4:Unit 4.18
Acknowledgements
We thank all of the individuals who have worked on polysome methods in the J.B.-S. group in the past, especially Cristina Branco-Price, Sheila Fennoy, Riki Kawaguchi, Angelika Mustroph, Reed Sorenson, Joanna Werner-Fraczek, Alan Williams, and Eugenia Zanetti and Nicholas Ingolia for helpful discussions on rRNA subtraction and cloning ribosome-protected fragments. This work was supported by the US National Science Foundation (IOS-0750811 to J. B.-S.) and MCB-1021969 (to J.B.-S.). J.B. was funded by Marie Curie European Economic Community Fellowship PIOF-GA-2012-327954.
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Juntawong, P., Hummel, M., Bazin, J., Bailey-Serres, J. (2015). Ribosome Profiling: A Tool for Quantitative Evaluation of Dynamics in mRNA Translation. In: Alonso, J., Stepanova, A. (eds) Plant Functional Genomics. Methods in Molecular Biology, vol 1284. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2444-8_7
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DOI: https://doi.org/10.1007/978-1-4939-2444-8_7
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