Abstract
The functional roles of RNAs are often regulated by their structure. Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE) and dimethyl sulfate (DMS) base reactivity can be employed to investigate the flexibility of nucleotides and correlate it to the constraints imparted by base-pairing and/or protein-binding. In vivo, a multitude of proteins could bind an RNA molecule, regulating its structure and function. Hence, to obtain a more comprehensive view of the RNA structure–function relationship in vivo, it may be required to characterize both the RNA structure and the RNA-protein interaction network. In this chapter, we describe methods for characterizing the in vivo nucleotide flexibility of RNA in cells by SHAPE-MaP (SHAPE by Mutational Profiling) and DMS-MaP. In another chapter, we will discuss the characterization of RNA-protein interaction network by RNP-MaP.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Spitale RC, Flynn RA, Zhang QC, Crisalli P, Lee B, Jung JW, Kuchelmeister HY, Batista PJ, Torre EA, Kool ET, Chang HY (2015) Structural imprints in vivo decode RNA regulatory mechanisms. Nature 519:486–490
Ding Y, Tang Y, Kwok CK, Zhang Y, Bevilacqua PC, Assmann SM (2014) In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features. Nature 505:696–700
Rouskin S, Zubradt M, Washietl S, Kellis M, Weissman JS (2014) Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature 505(7485):701–705. https://doi.org/10.1038/nature12894
Saha K, England W, Fernandez MM, Biswas T, Spitale RC, Ghosh G (2020) Structural disruption of exonic stem–loops immediately upstream of the intron regulates mammalian splicing. Nucleic Acids Res 48(11):6294–6309. https://doi.org/10.1093/nar/gkaa358
Saha K, Fernandez MM, Biswas T, Joseph S, Ghosh G (2021) Discovery of a pre-mRNA structural scaffold as a contributor to the mammalian splicing code. Nucleic Acids Res 49(12):7103–7121. https://doi.org/10.1093/nar/gkab533
Mustoe AM, Busan S, Rice GM, Hajdin CE, Peterson BK, Ruda VM, Kubica N, Nutiu R, Baryza JL, Weeks KM (2018) Pervasive regulatory functions of mRNA structure revealed by high-resolution SHAPE probing. Cell 173(1):181–195.e118. https://doi.org/10.1016/j.cell.2018.02.034
Cléry A, Krepl M, Nguyen CKX, Moursy A, Jorjani H, Katsantoni M, Okoniewski M, Mittal N, Zavolan M, Sponer J, Allain FH (2021) Structure of SRSF1 RRM1 bound to RNA reveals an unexpected bimodal mode of interaction and explains its involvement in SMN1 exon7 splicing. Nat Commun 12(1):428. https://doi.org/10.1038/s41467-020-20481-w
Saldi T, Riemondy K, Erickson B, Bentley DL (2021) Alternative RNA structures formed during transcription depend on elongation rate and modify RNA processing. Mol Cell 81(8):1789–1801.e1785. https://doi.org/10.1016/j.molcel.2021.01.040
Busan S, Weidmann CA, Sengupta A, Weeks KM (2019) Guidelines for SHAPE reagent choice and detection strategy for RNA structure probing studies. Biochemistry 58(23):2655–2664. https://doi.org/10.1021/acs.biochem.8b01218
Marinus T, Fessler AB, Ogle CA, Incarnato D (2021) A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy. Nucleic Acids Res 49(6):e34. https://doi.org/10.1093/nar/gkaa1255
Tomezsko PJ, Corbin VDA, Gupta P, Swaminathan H, Glasgow M, Persad S, Edwards MD, McIntosh L, Papenfuss AT, Emery A, Swanstrom R, Zang T, Lan TCT, Bieniasz P, Kuritzkes DR, Tsibris A, Rouskin S (2020) Determination of RNA structural diversity and its role in HIV-1 RNA splicing. Nature 582(7812):438–442. https://doi.org/10.1038/s41586-020-2253-5
Mustoe AM, Lama NN, Irving PS, Olson SW, Weeks KM (2019) RNA base-pairing complexity in living cells visualized by correlated chemical probing. Proc Natl Acad Sci U S A 116(49):24574–24582. https://doi.org/10.1073/pnas.1905491116
Smola MJ, Weeks KM (2018) In-cell RNA structure probing with SHAPE-MaP. Nat Protoc 13(6):1181–1195. https://doi.org/10.1038/nprot.2018.010
McGinnis JL, Dunkle JA, Cate JH, Weeks KM (2012) The mechanisms of RNA SHAPE chemistry. J Am Chem Soc 134(15):6617–6624. https://doi.org/10.1021/ja2104075
Barker SL, LaRocca PJ (1994) Method of production and control of a commercial tissue culture surface. J Tissue Cult Methods 16(3):151–153. https://doi.org/10.1007/BF01540642
Zubradt M, Gupta P, Persad S, Lambowitz AM, Weissman JS, Rouskin S (2017) DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Nat Methods 14:75–82
Busan S, Weeks KM (2018) Accurate detection of chemical modifications in RNA by mutational profiling (MaP) with ShapeMapper 2. RNA 24(2):143–148. https://doi.org/10.1261/rna.061945.117
Siegfried NA, Busan S, Rice GM, Nelson JA, Weeks KM (2014) RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). Nat Methods 11(9):959–965. https://doi.org/10.1038/nmeth.3029
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Saha, K., Ghosh, G. (2023). Chemical Probing of RNA Structure In Vivo Using SHAPE-MaP and DMS-MaP. In: Lin, RJ. (eds) RNA-Protein Complexes and Interactions. Methods in Molecular Biology, vol 2666. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3191-1_6
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3191-1_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3190-4
Online ISBN: 978-1-0716-3191-1
eBook Packages: Springer Protocols