Keth-seq for transcriptome-wide RNA structure mapping

Abstract

RNA secondary structure is critical to RNA regulation and function. We report a new N3-kethoxal reagent that allows fast and reversible labeling of single-stranded guanine bases in live cells. This N3-kethoxal-based chemistry allows efficient RNA labeling under mild conditions and transcriptome-wide RNA secondary structure mapping.

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Fig. 1: N3-kethoxal and experimental evaluation of its selectivity, cell permeability and reversibility.
Fig. 2: Keth-seq method and the profile around rG4 regions.

Data availability

All genomic data sets have been deposited in the Gene Expression Omnibus under accession number GSE122096. Other data and materials are available from the authors upon reasonable request.

Code availability

All custom codes used in this study are available at https://github.com/Tsinghua-gongjing/Keth-seq.

References

  1. 1.

    Wan, Y., Kertesz, M., Spitale, R. C., Segal, E. & Chang, H. Y. Understanding the transcriptome through RNA structure. Nat. Rev. Genet. 12, 641–655 (2011).

    CAS  Article  Google Scholar 

  2. 2.

    Kubota, M., Tran, C. & Spitale, R. C. Progress and challenges for chemical probing of RNA structure inside living cells. Nat. Chem. Biol. 11, 933–941 (2015).

    CAS  Article  Google Scholar 

  3. 3.

    Kertesz, M. et al. Genome-wide measurement of RNA secondary structure in yeast. Nature 467, 103–107 (2010).

    CAS  Article  Google Scholar 

  4. 4.

    Underwood, J. G. et al. FragSeq: transcriptome-wide RNA structure probing using high-throughput sequencing. Nat. Methods 7, 995–1001 (2010).

    CAS  Article  Google Scholar 

  5. 5.

    Lucks, J. B. et al. Multiplexed RNA structure characterization with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Proc. Natl Acad. Sci. USA 108, 11063–11068 (2011).

    CAS  Article  Google Scholar 

  6. 6.

    Rouskin, S., Zubradt, M., Washietl, S., Kellis, M. & Weissman, J. S. Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature 505, 701–705 (2014).

    CAS  Article  Google Scholar 

  7. 7.

    Ding, Y. et al. In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features. Nature 505, 696–700 (2014).

    CAS  Article  Google Scholar 

  8. 8.

    Talkish, J., May, G., Lin, Y., Woolford, J. L. & McManus, C. J. Mod-seq: high-throughput sequencing for chemical probing of RNA structure. RNA 20, 713–720 (2014).

    CAS  Article  Google Scholar 

  9. 9.

    Wan, Y. et al. Landscape and variation of RNA secondary structure across the human transcriptome. Nature 505, 706–709 (2014).

    CAS  Article  Google Scholar 

  10. 10.

    Spitale, R. C. et al. Structural imprints in vivo decode RNA regulatory mechanisms. Nature 519, 486–490 (2015).

    CAS  Article  Google Scholar 

  11. 11.

    Zubradt, M. et al. DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Nat. Methods 14, 75–82 (2016).

    Article  Google Scholar 

  12. 12.

    Lu, Z. & Chang, H. Y. Decoding the RNA structurome. Curr. Opin. Struct. Biol. 36, 142–148 (2016).

    CAS  Article  Google Scholar 

  13. 13.

    National Toxicology Program. Dimethyl sulfate. Rep. Carcinog. 12, 174–175 (2011).

    Google Scholar 

  14. 14.

    Merino, E. J., Wilkinson, K. A., Coughlan, J. L. & Weeks, K. M. RNA structure analysis at single nucleotide resolution by selective 2'-hydroxyl acylation and primer extension (SHAPE). J. Am. Chem. Soc. 127, 4223–4231 (2005).

    CAS  Article  Google Scholar 

  15. 15.

    Mitchell, D. et al. Glyoxals as in vivo RNA structural probes of guanine base-pairing. RNA 24, 114–124 (2018).

    CAS  Article  Google Scholar 

  16. 16.

    Mitchell, D. et al. In vivo RNA structural probing of uracil and guanine base pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). RNA 25, 147–157 (2019).

    CAS  Article  Google Scholar 

  17. 17.

    Wang, P. Y., Sexton, A. N., Culligan, W. J. & Simon, M. D. Carbodiimide reagents for the chemical probing of RNA structure in cells. RNA 25, 135–146 (2019).

    CAS  Article  Google Scholar 

  18. 18.

    Feng, C. et al. Light-activated chemical probing of nucleobase solvent accessibility inside cells. Nat. Chem. Biol. 14, 276–283 (2018).

    CAS  Article  Google Scholar 

  19. 19.

    Xu, Z. & Culver, G.M. In Methods in Enzymology; Biophysical, Chemical, and Functional Probes of Rna Structure, Interactions and Folding, Pt A (ed. Herschalag, D.) Vol 468, 47–165 (Academic Press, 2009).

  20. 20.

    Morse, D. P. & Bass, B. L. Detection of inosine in messenger RNA by inosine-specific cleavage. Biochemistry 36, 8429–8434 (1997).

    CAS  Article  Google Scholar 

  21. 21.

    Andronescu, M., Bereg, V., Hoos, H. H. & Condon, A. RNA STRAND: the RNA secondary structure and statistical analysis database. BMC Bioinforma. 9, 340 (2008).

    Article  Google Scholar 

  22. 22.

    Kwok, C. K., Marsico, G., Sahakyan, A. B., Chambers, V. S. & Balasubramanian, S. rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome. Nat. Methods 13, 841–844 (2016).

    CAS  Article  Google Scholar 

  23. 23.

    Guo, J. U. & Bartel, D. P. RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria. Science 353, aaf5371 (2016).

    Article  Google Scholar 

  24. 24.

    Biffi, G., Di Antonio, M., Tannahill, D. & Balasubramanian, S. Visualization and selective chemical targeting of RNA G-quadruplex structures in the cytoplasm of human cells. Nat. Chem. 6, 75–80 (2014).

    CAS  Article  Google Scholar 

  25. 25.

    Kwok, C. K., Marsico, G. & Balasubramanian, S. Detecting RNA G-quadruplexes (rG4s) in the transcriptome. Cold Spring Harb. Perspect. Biol. 10, a032284 (2018).

    Article  Google Scholar 

  26. 26.

    Spitale, R. C. et al. RNA SHAPE analysis in living cells. Nat. Chem. Biol. 9, 18–20 (2013).

    CAS  Article  Google Scholar 

  27. 27.

    Lu, Z. et al. RNA Duplex map in living cells reveals higher-order transcriptome structure. Cell 165, 1267–1279 (2016).

    CAS  Article  Google Scholar 

  28. 28.

    Kalvari, I. et al. Rfam 13.0: shifting to a genome-centric resource for non-coding RNA families. Nucleic Acids Res. 46, D335–D442 (2018).

    CAS  Article  Google Scholar 

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant nos. 21572172, 21778040 and 21822704 to X.W.; 21432008, 91753201 and 21721005 to X.Z.; 31671355, 91740204 and 31761163007 to Q.C.Z.) and National Institutes of Health grant no. HG008935 (C.H.). C.H. is an investigator of the Howard Hughes Medical Institute. X.W. was supported by China Scholarship Council (CSC) during his visit to the University of Chicago. We acknowledge S. Frank, who edited the manuscript.

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X.W., Q.C.Z., X.Z. and C.H. conceived the project, designed the experiments and wrote the manuscript. X.W., Y.C. and T.W. performed the experiments with the help of F.W., S.Y., Y.Y., G.L., K.C., L.H., H.M. and P.W. J.G. and Q.C.Z. designed and performed the bioinformatics analysis.

Corresponding authors

Correspondence to Qiangfeng Cliff Zhang or Xiang Zhou or Chuan He.

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Competing interests

C.H. is a scientific founder and a member of the scientific advisory board of Accent Therapeutics, Inc., and a shareholder of Epican Genetech.

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Supplementary Figs. 1–15 and Notes 1 and 2.

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Weng, X., Gong, J., Chen, Y. et al. Keth-seq for transcriptome-wide RNA structure mapping. Nat Chem Biol 16, 489–492 (2020). https://doi.org/10.1038/s41589-019-0459-3

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