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Probing N 6-methyladenosine (m6A) RNA Modification in Total RNA with SCARLET

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Post-Transcriptional Gene Regulation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1358))

Abstract

Posttranscriptional N 6-methyladenosine (m6A) RNA modification is indispensable for cell development and viability; however, functional investigation of m6A biological function has been hindered by the lack of methods for its precise identification and quantitation. Here, we describe a method that accurately identifies m6A position and modification fraction in human messenger RNA (mRNA) and long noncoding RNA (lncRNA) at single-nucleotide resolution, termed as “site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET)” (Fig. 1). This method combines two previously established techniques, site-specific cleavage and splint ligation, to probe the m6A RNA modification status at any mRNA/lncRNA site in the total RNA pool.

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Acknowledgments

We thank Dr. Q. Dai for assistance with chemical synthesis, and Dr. M. Parisien for bioinformatic analysis to choose potential target nucleotide sites from mRNA /lncRNA . This work was supported by a NIH grant (GM088599 to T.P. and C.H.).

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Authors and Affiliations

  1. Department of Chemistry, University of Chicago, Chicago, IL, 60637, USA

    Nian Liu

  2. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, 60637, USA

    Tao Pan

  3. Institute of Biophysical Dynamics, University of Chicago, Chicago, IL, 60637, USA

    Tao Pan

Authors
  1. Nian Liu
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  2. Tao Pan
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Corresponding author

Correspondence to Tao Pan .

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Editors and Affiliations

  1. Laboratory of Translational Genomics, Centre for Integrative Biology, University of Trento, Mattarello, Trento, Italy

    Erik Dassi

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Liu, N., Pan, T. (2016). Probing N 6-methyladenosine (m6A) RNA Modification in Total RNA with SCARLET. In: Dassi, E. (eds) Post-Transcriptional Gene Regulation. Methods in Molecular Biology, vol 1358. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3067-8_17

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  • DOI: https://doi.org/10.1007/978-1-4939-3067-8_17

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3066-1

  • Online ISBN: 978-1-4939-3067-8

  • eBook Packages: Springer Protocols

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