Deoxyribozyme-based Method for Absolute Quantification of -methyladenosine Fractions at Specific Sites of RNA

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2020 Apr 10

PMID 32269077

Citations 5

Authors

Magda Bujnowska

Jiacheng Zhang

Qing Dai

Emily M Heideman

Jingyi Fei

Affiliations

Soon will be listed here.

Abstract

-Methyladenosine (mA) is the most prevalent modified base in eukaryotic mRNA and long noncoding RNA. Although candidate sites for the mA modification are identified at the transcriptomic level, methods for site-specific quantification of absolute mA modification levels are still limited. Herein, we present a facile method implementing a deoxyribozyme, VMC10, which preferentially cleaves the unmodified RNA. We leveraged reverse transcription and real-time quantitative PCR along with key control experiments to quantify the methylation fraction of specific mA sites. We validated the accuracy of this method with synthetic RNA in which methylation fractions ranged from 0 to 100% and applied our method to several endogenous sites that were previously identified in sequencing-based studies. This method provides a time- and cost-effective approach for absolute quantification of the mA fraction at specific loci, with the potential for multiplexed quantifications, expanding the current toolkit for studying RNA modifications.

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References

Sednev M, Mykhailiuk V, Choudhury P, Halang J, Sloan K, Bohnsack M . N -Methyladenosine-Sensitive RNA-Cleaving Deoxyribozymes. Angew Chem Int Ed Engl. 2018; 57(46):15117-15121. DOI: 10.1002/anie.201808745. View

Kasowitz S, Ma J, Anderson S, Leu N, Xu Y, Gregory B . Nuclear m6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development. PLoS Genet. 2018; 14(5):e1007412. PMC: 5991768. DOI: 10.1371/journal.pgen.1007412. View

Shi H, Wei J, He C . Where, When, and How: Context-Dependent Functions of RNA Methylation Writers, Readers, and Erasers. Mol Cell. 2019; 74(4):640-650. PMC: 6527355. DOI: 10.1016/j.molcel.2019.04.025. View

Lei Z, Yi C . A Radiolabeling-Free, qPCR-Based Method for Locus-Specific Pseudouridine Detection. Angew Chem Int Ed Engl. 2017; 56(47):14878-14882. DOI: 10.1002/anie.201708276. View

Tserovski L, Marchand V, Hauenschild R, Blanloeil-Oillo F, Helm M, Motorin Y . High-throughput sequencing for 1-methyladenosine (m(1)A) mapping in RNA. Methods. 2016; 107:110-21. DOI: 10.1016/j.ymeth.2016.02.012. View

Nachtergaele S, He C . Chemical Modifications in the Life of an mRNA Transcript. Annu Rev Genet. 2018; 52:349-372. PMC: 6436393. DOI: 10.1146/annurev-genet-120417-031522. View

Warda A, Kretschmer J, Hackert P, Lenz C, Urlaub H, Hobartner C . Human METTL16 is a -methyladenosine (mA) methyltransferase that targets pre-mRNAs and various non-coding RNAs. EMBO Rep. 2017; 18(11):2004-2014. PMC: 5666602. DOI: 10.15252/embr.201744940. View

Harcourt E, Ehrenschwender T, Batista P, Chang H, Kool E . Identification of a selective polymerase enables detection of N(6)-methyladenosine in RNA. J Am Chem Soc. 2013; 135(51):19079-82. PMC: 3905807. DOI: 10.1021/ja4105792. View

Zheng G, Dahl J, Niu Y, Fedorcsak P, Huang C, Li C . ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell. 2012; 49(1):18-29. PMC: 3646334. DOI: 10.1016/j.molcel.2012.10.015. View

10.

Santoro S, Joyce G . A general purpose RNA-cleaving DNA enzyme. Proc Natl Acad Sci U S A. 1997; 94(9):4262-6. PMC: 20710. DOI: 10.1073/pnas.94.9.4262. View

11.

Liu N, Parisien M, Dai Q, Zheng G, He C, Pan T . Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA. RNA. 2013; 19(12):1848-56. PMC: 3884656. DOI: 10.1261/rna.041178.113. View

12.

Maity A, Das B . N6-methyladenosine modification in mRNA: machinery, function and implications for health and diseases. FEBS J. 2015; 283(9):1607-30. DOI: 10.1111/febs.13614. View

13.

Linder B, Grozhik A, Olarerin-George A, Meydan C, Mason C, Jaffrey S . Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat Methods. 2015; 12(8):767-72. PMC: 4487409. DOI: 10.1038/nmeth.3453. View

14.

Zhang Z, Chen L, Zhao Y, Yang C, Roundtree I, Zhang Z . Single-base mapping of mA by an antibody-independent method. Sci Adv. 2019; 5(7):eaax0250. PMC: 6609220. DOI: 10.1126/sciadv.aax0250. View

15.

Garcia-Campos M, Edelheit S, Toth U, Safra M, Shachar R, Viukov S . Deciphering the "mA Code" via Antibody-Independent Quantitative Profiling. Cell. 2019; 178(3):731-747.e16. DOI: 10.1016/j.cell.2019.06.013. View

16.

Potapov V, Ong J, Langhorst B, Bilotti K, Cahoon D, Canton B . A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining. Nucleic Acids Res. 2018; 46(13):e79. PMC: 6061786. DOI: 10.1093/nar/gky303. View

17.

Ke S, Pandya-Jones A, Saito Y, Fak J, Vagbo C, Geula S . mA mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover. Genes Dev. 2017; 31(10):990-1006. PMC: 5495127. DOI: 10.1101/gad.301036.117. View

18.

Boccaletto P, Machnicka M, Purta E, Piatkowski P, Baginski B, Wirecki T . MODOMICS: a database of RNA modification pathways. 2017 update. Nucleic Acids Res. 2017; 46(D1):D303-D307. PMC: 5753262. DOI: 10.1093/nar/gkx1030. View

19.

Xiao Y, Wang Y, Tang Q, Wei L, Zhang X, Jia G . An Elongation- and Ligation-Based qPCR Amplification Method for the Radiolabeling-Free Detection of Locus-Specific N -Methyladenosine Modification. Angew Chem Int Ed Engl. 2018; 57(49):15995-16000. DOI: 10.1002/anie.201807942. View

20.

Dai Q, Fong R, Saikia M, Stephenson D, Yu Y, Pan T . Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine. Nucleic Acids Res. 2007; 35(18):6322-9. PMC: 2094055. DOI: 10.1093/nar/gkm657. View