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Genome-wide quantification of 5′-phosphorylated mRNA degradation intermediates for analysis of ribosome dynamics

Nature Protocols volume 11, pages 359–376 (2016)Cite this article

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Abstract

Co-translational mRNA degradation is a widespread process in which 5′–3′ exonucleolytic degradation follows the last translating ribosome, thus producing an in vivo ribosomal footprint that delimits the 5′ position of the mRNA molecule within the ribosome. To study this degradation process and ribosome dynamics, we developed 5PSeq, which is a method that profiles the genome-wide abundance of mRNA degradation intermediates by virtue of their 5′-phosphorylated (5′P) ends. The approach involves targeted ligation of an oligonucleotide to the 5′P end of mRNA degradation intermediates, followed by depletion of rRNA molecules, reverse transcription of 5′P mRNAs and Illumina high-throughput sequencing. 5PSeq can identify translational pauses at rare codons that are often masked when using alternative methods. This approach can be applied to previously extracted RNA samples, and it is straightforward and does not require polyribosome purification or in vitro RNA footprinting. The protocol we describe here can be applied to Saccharomyces cerevisiae and potentially to other eukaryotic organisms. Three days are required to generate 5PSeq libraries.

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Figure 1: Detailed 5PSeq experimental workflow.
Figure 2: 5PSeq quality controls and anticipated results.
Figure 3: Schematic of alternative 5′ RNA treatments.
Figure 4: Comparison of alternative decapping enzymes.

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Acknowledgements

We thank A. Jones for help in editing the manuscript. We thank the members of the Steinmetz laboratory for helpful discussions and critical comments. We thank M. Livingstone for access to alternative decapping enzymes. This study was technically supported by the European Molecular Biology Laboratory (EMBL) Genomics Core Facility. This study was financially supported by the US National Institutes of Health (NIH; grants R01 GM068717 and P01 HG000205), Deutsche Forschungsgemeinschaft (1422/3-1) and a European Research Council Advanced Investigator Grant (no. AdG-294542) to L.M.S.

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Authors and Affiliations

  1. European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany

    Vicent Pelechano & Lars M Steinmetz

  2. Stanford Genome Technology Center, Palo Alto, California, USA

    Wu Wei & Lars M Steinmetz

  3. Department of Genetics, Stanford University School of Medicine, Stanford, California, USA

    Wu Wei & Lars M Steinmetz

Authors
  1. Vicent Pelechano
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  2. Wu Wei
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Contributions

V.P., W.W. and L.M.S. conceived the project. V.P. developed the 5PSeq method. W.W. and V.P. performed data analysis. L.M.S. supervised the study. All authors wrote the manuscript.

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Correspondence to Lars M Steinmetz.

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Pelechano, V., Wei, W. & Steinmetz, L. Genome-wide quantification of 5′-phosphorylated mRNA degradation intermediates for analysis of ribosome dynamics. Nat Protoc 11, 359–376 (2016). https://doi.org/10.1038/nprot.2016.026

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