Abstract
Methylation of RNA is normally monitored in purified cell lysates using next-generation sequencing, gel electrophoresis, or mass spectrometry as readouts. These bulk methods require the RNA from ~104 to 107 cells to be pooled to generate sufficient material for analysis. Here we describe a method—methylation-sensitive RNA in situ hybridization (MR-FISH)—that assays rRNA methylation in bacteria on a cell-by-cell basis, using methylation-sensitive hybridization probes and fluorescence microscopy. We outline step-by-step protocols for designing probes, in situ hybridization, and analysis of data using freely available code.
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
Adler M, Weissmann B, Gutman AB (1958) Occurrence of methylated purine bases in yeast ribonucleic acid. J Biol Chem 230:717–723
Starr JL, Fefferman R (1964) The occurrence of methylated bases in ribosomal ribonucleic acid of Escherichia coli K12 W-6. J Biol Chem 239:3457–3461
Kellner S, Burhenne J, Helm M (2010) Detection of RNA modifications. RNA Biol 7:237–247
Helm M, Motorin Y (2017) Detecting RNA modifications in the epitranscriptome: predict and validate. Nat Rev Genet 18:275–291. https://doi.org/10.1038/nrg.2016.169
Motorin Y, Muller S, Behm-Ansmant I, Branlant C (2007) Identification of modified residues in RNAs by reverse transcription-based methods. Methods Enzymol 425:21–53. https://doi.org/10.1016/S0076-6879(07)25002-5
Dominissini D, Moshitch-Moshkovitz S, Salmon-Divon M et al (2013) Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing. Nat Protoc 8:176–189. https://doi.org/10.1038/nprot.2012.148
Ovcharenko A, Rentmeister A (2018) Emerging approaches for detection of methylation sites in RNA. Open Biol 8:180121. https://doi.org/10.1098/rsob.180121
Ranasinghe RT, Challand MR, Ganzinger KA et al (2018) Detecting RNA base methylations in single cells by in situ hybridization. Nat Commun 9. https://doi.org/10.1038/s41467-017-02714-7
Dennis PP, Bremer H (2008) Modulation of chemical composition and other parameters of the cell at different exponential growth rates. EcoSal Plus 3. https://doi.org/10.1128/ecosal.5.2.3
Micura R, Pils W, Höbartner C et al (2001) Methylation of the nucleobases in RNA oligonucleotides mediates duplex-hairpin conversion. Nucleic Acids Res 29:3997–4005
Roost C, Lynch SR, Batista PJ et al (2015) Structure and thermodynamics of N 6-Methyladenosine in RNA: a spring-Loaded Base modification. J Am Chem Soc 137:2107–2115. https://doi.org/10.1021/ja513080v
Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14:303–308. https://doi.org/10.1038/nbt0396-303
Bonnet G, Tyagi S (1999) Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc Natl Acad Sci U S A 96:6171–6176
Marras SAE, Kramer FR, Tyagi S (2002) Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res 30:e122
Markham NR, Zuker M (2005) DINAMelt web server for nucleic acid melting prediction. Nucleic Acids Res 33:577–581. https://doi.org/10.1093/nar/gki591
Markham NR, Zuker M (2008) UNAFold: software for nucleic acid folding and hybridization. Methods Mol Biol 453:3–31. https://doi.org/10.1007/978-1-60327-429-6_1
Fuchs BM, Glockner FO, Wulf J, Amann R (2000) Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 66:3603–3607. https://doi.org/10.1128/AEM.66.8.3603-3607.2000
Fuchs BM, Wallner G, Beisker W et al (1998) Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 64:4973–4982. https://doi.org/10.1007/s00214-011-0990-0
Fuchs BM, Syutsubo K, Ludwig W, Amann R (2001) In situ accessibility of Escherichia coli 23S rRNA to fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 67:961–968. https://doi.org/10.1128/AEM.67.2.961-968.2001
Rueden CT, Schindelin J, Hiner MC et al (2017) ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18:1–26. https://doi.org/10.1186/s12859-017-1934-z
Ranasinghe RT, Challand MR, Ganzinger KA et al (2017) Detecting RNA base methylations in single cells by in situ hybridization (datasets). https://doi.org/10.6084/m9.figshare.4667959.v1
Acknowledgments
This work was supported by the EU Innovative Medicines Initiative, IMI (RAPP-ID project, grant agreement, no. 115153), the UK Biotechnology and Biological Sciences Research Council, BBSRC (Project Grant: BB/J017906/1), and the UK Engineering and Physical Sciences Research Council, EPRSC (Project Grant: EP/M027546/1). D.K. is supported by the Royal Society.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Ganzinger, K.A., Challand, M.R., Spencer, J., Klenerman, D., Ranasinghe, R.T. (2019). Imaging rRNA Methylation in Bacteria by MR-FISH. In: Shav-Tal, Y. (eds) Imaging Gene Expression. Methods in Molecular Biology, vol 2038. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9674-2_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9674-2_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9673-5
Online ISBN: 978-1-4939-9674-2
eBook Packages: Springer Protocols