Abstract
Current development of epitranscriptomics field requires efficient experimental protocols for precise mapping and quantification of various modified nucleotides in RNA. Despite important advances in the field during the last 10 years, this task is still extremely laborious and time-consuming, even when high-throughput analytical approaches are employed. Moreover, only a very limited subset of RNA modifications can be detected and only rarely be quantified by these powerful techniques. In the past, we developed and successfully applied alkaline fragmentation-based RiboMethSeq approach for mapping and precise quantification of multiple 2′-O-methylation residues in ribosomal RNA. Here we describe a RiboMethSeq protocol adapted for the analysis of bacterial and eukaryotic tRNA species, which also contain 2′-O-methylations at functionally important RNA regions.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Helm M, Alfonzo JD (2014) Posttranscriptional RNA modifications: playing metabolic games in a cell’s chemical Legoland. Chem Biol 21(2):174–185
Helm M, Motorin Y (2017) Detecting RNA modifications in the epitranscriptome: predict and validate. Nat Rev Genet 18(5):275–291
Rana AK, Ankri S (2016) Reviving the RNA world: an insight into the appearance of RNA methyltransferases. Front Genet 7:99
Towns WL, Begley TJ (2012) Transfer RNA methytransferases and their corresponding modifications in budding yeast and humans: activities, predications, and potential roles in human health. DNA Cell Biol 31(4):434–454
Motorin Y, Helm M (2011) RNA nucleotide methylation. Wiley Interdiscip Rev RNA 2(5):611–631
Gilbert WV, Bell TA, Schaening C (2016) Messenger RNA modifications: form, distribution, and function. Science 352(6292):1408–1412
Yue Y, Liu J, He C (2015) RNA N6-methyladenosine methylation in post-transcriptional gene expression regulation. Genes Dev 29(13):1343–1355
Sarin LP, Leidel SA (2014) Modify or die?—RNA modification defects in metazoans. RNA Biol 11(12):1555–1567
Maden BE (2000) Tetrahydrofolate and tetrahydromethanopterin compared: functionally distinct carriers in C1 metabolism. Biochem J 350(Pt 3):609–629
Maden BE (2001) Mapping 2′-O-methyl groups in ribosomal RNA. Methods 25(3):374–382
Yu YT, Shu MD, Steitz JA (1997) A new method for detecting sites of 2′-O-methylation in RNA molecules. RNA 3(3):324–331
Dong Z-W, Shao P, Diao L-T, Zhou H, Yu C-H, Qu L-H (2012) RTL-P: a sensitive approach for detecting sites of 2′-O-methylation in RNA molecules. Nucleic Acids Res 40(20):e157
Chan CTY, Dyavaiah M, DeMott MS, Taghizadeh K, Dedon PC, Begley TJ (2010) A quantitative systems approach reveals dynamic control of tRNA modifications during cellular stress. PLoS Genet [Internet] [cited 2017 May 17];6(12)
Marchand V, Ayadi L, El Hajj A, Blanloeil-Oillo F, Helm M, Motorin Y (2017) High-throughput mapping of 2′-O-Me residues in RNA using next-generation sequencing (illumina RiboMethSeq protocol). Methods Mol Biol 1562:171–187
Marchand V, Blanloeil-Oillo F, Helm M, Motorin Y (2016) Illumina-based RiboMethSeq approach for mapping of 2′-O-Me residues in RNA. Nucleic Acids Res 44(16):e135
Marchand V, Pichot F, Thüring K, Ayadi L, Freund I, Dalpke A et al (2017) Next-generation sequencing-based RiboMethSeq protocol for analysis of tRNA 2′-O-methylation. Biomolecules [Internet] [cited 2017 Jul 12];7(1)
Schmitt ME, Brown TA, Trumpower BL (1990) A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res 18(10):3091–3092
Wilfinger WW, Mackey K, Chomczynski P (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques 22(3):474–476, 478–481
Acknowledgement
This work was supported by joint ANR-DFG grant HTRNAMod (ANR-13-ISV8-0001/HE 3397/8-1) to MH and YM, and AO Lorraine University-Lorraine Region “Aberrant RNA methylation in cancer” funding to YM.
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
Galvanin, A., Ayadi, L., Helm, M., Motorin, Y., Marchand, V. (2019). Mapping and Quantification of tRNA 2′-O-Methylation by RiboMethSeq. In: Wajapeyee, N., Gupta, R. (eds) Epitranscriptomics. Methods in Molecular Biology, vol 1870. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8808-2_21
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8808-2_21
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8807-5
Online ISBN: 978-1-4939-8808-2
eBook Packages: Springer Protocols