Abstract
This protocol is an extension to: Nat. Protoc. 10, 1643–1669 (2015); doi:10.1038/nprot.2015.103; published online 01 October 2015
RNAs play key roles in many cellular processes. The underlying structure of RNA is an important determinant of how transcripts function, are processed, and interact with RNA-binding proteins and ligands. RNA structure analysis by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) takes advantage of the reactivity of small electrophilic chemical probes that react with the 2′-hydroxyl group to assess RNA structure at nucleotide resolution. When coupled with mutational profiling (MaP), in which modified nucleotides are detected as internal miscodings during reverse transcription and then read out by massively parallel sequencing, SHAPE yields quantitative per-nucleotide measurements of RNA structure. Here, we provide an extension to our previous in vitro SHAPE-MaP protocol with detailed guidance for undertaking and analyzing SHAPE-MaP probing experiments in live cells. The MaP strategy works for both abundant-transcriptome experiments and for cellular RNAs of low to moderate abundance, which are not well examined by whole-transcriptome methods. In-cell SHAPE-MaP, performed in roughly 3 d, can be applied in cell types ranging from bacteria to cultured mammalian cells and is compatible with a variety of structure-probing reagents. We detail several strategies by which in-cell SHAPE-MaP can inform new biological hypotheses and emphasize downstream analyses that reveal sequence or structure motifs important for RNA interactions in cells.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Sharp, P.A. The centrality of RNA. Cell 136, 577–580 (2009).
Wang, Z. & Burge, C.B. Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA 14, 802–813 (2008).
Ghildiyal, M. & Zamore, P.D. Small silencing RNAs: an expanding universe. Nat. Rev. Genet. 10, 94–108 (2009).
Sherwood, A.V. & Henkin, T.M. Riboswitch-mediated gene regulation: novel RNA architectures dictate gene expression responses. Annu. Rev. Microbiol. 70, 361–374 (2016).
Lau, M.W.L. & Ferré-D'Amaré, A.R. Many activities, one structure: functional plasticity of ribozyme folds. Molecules 21, 1570 (2016).
Rinn, J.L. & Chang, H.Y. Genome regulation by long noncoding RNAs. Annu. Rev. Biochem. 81, 145–166 (2012).
Engreitz, J.M., Ollikainen, N. & Guttman, M. Long non-coding RNAs: spatial amplifiers that control nuclear structure and gene expression. Nat. Rev. Mol. Cell Biol. 17, 756–770 (2016).
Cech, T.R. & Steitz, J.A. The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157, 77–94 (2014).
Mortimer, S.A., Kidwell, M.A. & Doudna, J.A. Insights into RNA structure and function from genome-wide studies. Nat. Rev. Genet. 15, 469–479 (2014).
Butcher, S.E. & Pyle, A.M. The molecular interactions that stabilize RNA tertiary structure: RNA motifs, patterns, and networks. Acc. Chem. Res. 44, 1302–1311 (2011).
Nicholson, B.L. & White, K.A. Exploring the architecture of viral RNA genomes. Curr. Opin. Virol. 12, 66–74 (2015).
Gebhard, L.G., Filomatori, C.V. & Gamarnik, A.V. Functional RNA elements in the dengue virus genome. Viruses 3, 1739–1756 (2011).
Licatalosi, D.D. & Darnell, R.B. RNA processing and its regulation: global insights into biological networks. Nat. Rev. Genet. 11, 75–87 (2010).
Spitale, R.C. et al. RNA SHAPE analysis in living cells. Nat. Chem. Biol. 9, 18–20 (2012).
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).
Loughrey, D., Watters, K.E., Settle, A.H. & Lucks, J.B. SHAPE-Seq 2.0: systematic optimization and extension of high-throughput chemical probing of RNA secondary structure with next generation sequencing. Nucleic Acids Res. 42, e165–e165 (2014).
Siegfried, N.A., Busan, S., Rice, G.M., Nelson, J.A.E. & Weeks, K.M. RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). Nat. Methods 11, 959–965 (2014).
Smola, M.J., Calabrese, J.M. & Weeks, K.M. Detection of RNA-protein interactions in living cells with SHAPE. Biochemistry 54, 6867–6875 (2015).
McGinnis, J.L. et al. In-cell SHAPE reveals that free 30S ribosome subunits are in the inactive state. Proc. Natl. Acad. Sci. USA 112, 2425–2430 (2015).
Tyrrell, J., McGinnis, J.L., Weeks, K.M. & Pielak, G.J. The cellular environment stabilizes adenine riboswitch RNA structure. Biochemistry 52, 8777–8785 (2013).
McGinnis, J.L. & Weeks, K.M. Ribosome RNA assembly intermediates visualized in living cells. Biochemistry 53, 3237–3247 (2014).
Fang, R., Moss, W.N., Rutenberg-Schoenberg, M. & Simon, M.D. Probing Xist RNA structure in cells using targeted structure-Seq. PLoS Genet 11, e1005668 (2015).
Smola, M.J., Rice, G.M., Busan, S., Siegfried, N.A. & Weeks, K.M. Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis. Nat. Protoc. 10, 1643–1669 (2015).
Smola, M.J. et al. SHAPE reveals transcript-wide interactions, complex structural domains, and protein interactions across the Xist lncRNA in living cells. Proc. Natl. Acad. Sci. USA 113, 10322–10327 (2016).
Weeks, K.M. & Mauger, D.M. Exploring RNA structural codes with SHAPE chemistry. Acc. Chem. Res. 44, 1280–1291 (2011).
Spitale, R.C. et al. Structural imprints in vivo decode RNA regulatory mechanisms. Nature 519, 486–490 (2015).
Homan, P.J. et al. Single-molecule correlated chemical probing of RNA. Proc. Natl. Acad. Sci. USA 111, 13858–13863 (2014).
Larman, B.C., Dethoff, E.A. & Weeks, K.M. Packaged and free satellite tobacco mosaic virus (STMV) RNA genomes adopt distinct conformational states. Biochemistry 56, 2175–2183 (2017).
Underwood, J.G. et al. FragSeq: transcriptome-wide RNA structure probing using high-throughput sequencing. Nat. Methods 7, 995–1001 (2010).
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).
Ding, Y. et al. In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features. Nature 505, 696–700 (2014).
Incarnato, D., Neri, F., Anselmi, F. & Oliviero, S. Genome-wide profiling of mouse RNA secondary structures reveals key features of the mammalian transcriptome. Genome Biol. 15, 491 (2014).
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 (2015).
Krokhotin, A., Mustoe, A.M., Weeks, K.M. & Dokholyan, N.V. Direct identification of base-paired RNA nucleotides by correlated chemical probing. RNA 23, 6–13 (2017).
McGinnis, J.L., Dunkle, J.A., Cate, J.H.D. & Weeks, K.M. The mechanisms of RNA SHAPE chemistry. J. Am. Chem. Soc. 134, 6617–6624 (2012).
Gherghe, C.M., Shajani, Z., Wilkinson, K.A., Varani, G. & Weeks, K.M. Strong correlation between SHAPE chemistry and the generalized NMR order parameter (S2) in RNA. J. Am. Chem. Soc. 130, 12244–12245 (2008).
Deigan, K.E., Li, T.W., Mathews, D.H. & Weeks, K.M. Accurate SHAPE-directed RNA structure determination. Proc. Natl. Acad. Sci. USA 106, 97–102 (2009).
Hajdin, C.E. et al. Accurate SHAPE-directed RNA secondary structure modeling, including pseudoknots. Proc. Natl. Acad. Sci. USA 110, 5498–5503 (2013).
Watters, K.E., Abbott, T.R. & Lucks, J.B. Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq. Nucleic Acids Res. 44, e12 (2016).
Takahashi, M.K. et al. Using in-cell SHAPE-Seq and simulations to probe structure-function design principles of RNA transcriptional regulators. RNA 22, 920–933 (2016).
Kuhlmann, M.M., Chattopadhyay, M., Stupina, V.A., Gao, F. & Simon, A.E. An RNA element that facilitates programmed ribosomal readthrough in turnip crinkle virus adopts multiple conformations. J. Virol. 90, 8575–8591 (2016).
Sztuba-Solinska, J. et al. Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA: a structural scaffold for nuclear, cytoplasmic and viral proteins. Nucleic Acids Res. 45, 6805–6821 (2017).
Lee, B. et al. Comparison of SHAPE reagents for mapping RNA structures inside living cells. RNA 23, 169–174 (2017).
Lavender, C.A. et al. Model-Free RNA sequence and structure alignment informed by SHAPE probing reveals a conserved alternate secondary structure for 16S rRNA. PLoS Comput. Biol. 11, e1004126 (2015).
Engreitz, J.M. et al. The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome. Science 341, 1237973–1237973 (2013).
Simon, M.D. et al. High-resolution Xist binding maps reveal two-step spreading during X-chromosome inactivation. Nature 504, 465–469 (2013).
Jabara, C.B., Jones, C.D., Roach, J., Anderson, J.A. & Swanstrom, R. Accurate sampling and deep sequencing of the HIV-1 protease gene using a primer ID. Proc. Natl. Acad. Sci. USA 108, 20166–20171 (2011).
Busan, S. & Weeks, K.M. Accurate detection of chemical modifications in RNA by mutational profiling (MaP) with ShapeMapper 2. RNA 24, 143–148 (2017).
Mortimer, S.A. & Weeks, K.M. A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry. J. Am. Chem. Soc. 129, 4144–4145 (2007).
Steen, K.-A., Siegfried, N.A. & Weeks, K.M. Synthesis of 1-methyl-7-nitroisatoic anhydride (1M7). Protoc. Exchange http://dx.doi.org/10.1038/protex.2011.255 (2011).
Turner, R., Shefer, K. & Ares, M. Safer one-pot synthesis of the 'SHAPE' reagent 1-methyl-7-nitroisatoic anhydride (1m7). RNA 19, 1857–1863 (2013).
Wang, X., Schwartz, J.C. & Cech, T.R. Nucleic acid-binding specificity of human FUS protein. Nucleic Acids Res. 43, 7535–7543 (2015).
Lagier-Tourenne, C., Polymenidou, M. & Cleveland, D.W. TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration. Hum. Mol. Genet. 19, R46–R64 (2010).
Bhardwaj, A., Myers, M.P., Buratti, E. & Baralle, F.E. Characterizing TDP-43 interaction with its RNA targets. Nucleic Acids Res. 41, 5062–5074 (2013).
Kondo, Y., Oubridge, C., van Roon, A.-M.M. & Nagai, K. Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site recognition. Elife 4 04986 (2015).
Acknowledgements
Work in our lab focused on developing quantitative and biophysically rigorous RNA structure-probing technologies is supported by the National Institutes of Health (NIH; R35 GM122532 and R01 AI068462). M.J.S. was a Graduate Research Fellow of the National Science Foundation (DGE-1144081) and was supported in part by an NIH training grant in molecular and cellular biophysics (T32 GM08570). We are indebted to the Calabrese laboratory at the University of North Carolina at Chapel Hill for assistance in developing in-cell probing strategies and to members of the Weeks laboratory for thoughtful feedback regarding the analysis algorithms and strategies described here.
Author information
Authors and Affiliations
Contributions
M.J.S. and K.M.W. conceived the use of SHAPE-MaP in living cells. M.J.S. developed the in-cell probing strategy and created the ΔSHAPE analysis procedure and software. Both authors wrote and edited the manuscript.
Corresponding author
Ethics declarations
Competing interests
K.M.W. has equity ownership in and serves as an advisor to Ribometrix, to which SHAPE-MaP technologies have been licensed. M.J.S. is an employee of Ribometrix.
Rights and permissions
About this article
Cite this article
Smola, M., Weeks, K. In-cell RNA structure probing with SHAPE-MaP. Nat Protoc 13, 1181–1195 (2018). https://doi.org/10.1038/nprot.2018.010
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2018.010
This article is cited by
-
Single-cell probing of RNA structure
Nature Methods (2024)
-
Pervasive downstream RNA hairpins dynamically dictate start-codon selection
Nature (2023)
-
Identifying the structures of individual RNA isoforms inside cells
Nature Methods (2023)
-
The p53 endoplasmic reticulum stress-response pathway evolved in humans but not in mice via PERK-regulated p53 mRNA structures
Cell Death & Differentiation (2023)
-
In vivo secondary structural analysis of Influenza A virus genomic RNA
Cellular and Molecular Life Sciences (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.