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Ribosome Profiling: A Tool for Quantitative Evaluation of Dynamics in mRNA Translation

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Plant Functional Genomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1284))

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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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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Authors and Affiliations

  1. Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, CA, 92521, USA

    Piyada Juntawong, Maureen Hummel, Jeremie Bazin & Julia Bailey-Serres

  2. Department of Genetics, Faculty of Science, Kasetsart University, Bangkok, 10900, Thailand

    Piyada Juntawong

  3. Saclay Plant Science, Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, Gif-sur-Yvette, 91198, France

    Jeremie Bazin

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  1. Piyada Juntawong
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  2. Maureen Hummel
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  3. Jeremie Bazin
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  4. Julia Bailey-Serres
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Correspondence to Julia Bailey-Serres .

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Editors and Affiliations

  1. Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA

    Jose M. Alonso

  2. Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA

    Anna N. Stepanova

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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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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2443-1

  • Online ISBN: 978-1-4939-2444-8

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