Advertisement

Methylated mRNA Nucleotides as Regulators for Ribosomal Translation

  • Thomas P. Hoernes
  • Matthias D. ErlacherEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1562)

Abstract

Methylated RNA nucleotides were recently discovered to be highly abundant in RNAs. The effects of these methylations were mainly attributed to altered mRNA stabilities, protein-binding affinities, or RNA structures. The direct impact of RNA modifications on the performance of the ribosome has not been investigated so far. In this chapter, we describe an approach that allows introducing RNA modifications site-specifically into coding sequences of mRNAs and determining their effect on the translation machinery in a well-defined bacterial in vitro system.

Key words

Splinted ligation In vitro translation RNA purification Peptide purification 

Notes

Acknowledgment

This work was supported by the FWF (P 22658-B12 and P 28494-BBL to M.E.)

References

  1. 1.
    El Yacoubi B, Bailly M, de Crecy-Lagard V (2012) Biosynthesis and function of posttranscriptional modifications of transfer RNAs. Annu Rev Genet 46:69–95CrossRefPubMedGoogle Scholar
  2. 2.
    Nedialkova DD, Leidel SA (2015) Optimization of codon translation rates via tRNA modifications maintains proteome integrity. Cell 161(7):1606–1618CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Wilson DN, Nierhaus KH (2007) The weird and wonderful world of bacterial ribosome regulation. Crit Rev Biochem Mol Biol 42(3):187–219CrossRefPubMedGoogle Scholar
  4. 4.
    Pan T (2013) N6-methyl-adenosine modification in messenger and long non-coding RNA. Trends Biochem Sci 38(4):204–209CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Edelheit S, Schwartz S, Mumbach MR, Wurtzel O, Sorek R (2013) Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs. PLoS Genet 9(6):e1003602CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ, Suter CM, Preiss T (2012) Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res 40(11):5023–5033CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Carlile TM, Rojas-Duran MF, Zinshteyn B, Shin H, Bartoli KM, Gilbert WV (2014) Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature 515:143–146CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lovejoy AF, Riordan DP, Brown PO (2014) Transcriptome-wide mapping of pseudouridines: pseudouridine synthases modify specific mRNAs in S. cerevisiae. PLoS One 9(10):e110799CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Hoernes TP, Erlacher MD (2016) Translating the epitranscriptome. WIREs RNA 2016. doi: 10.1002/wrna.1375
  10. 10.
    Li X, Xiong X, Wang K, Wang L, Shu X, Ma S, Yi C (2016) Transcriptome-wide mapping reveals reversible and dynamic N(1)-methyladenosine methylome. Nat Chem Biol 12(5):311–316CrossRefPubMedGoogle Scholar
  11. 11.
    Dominissini D, Nachtergaele S, Moshitch-Moshkovitz S, Peer E, Kol N, Ben-Haim MS, Dai Q, Di Segni A, Salmon-Divon M, Clark WC et al (2016) The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA. Nature 530(7591):441–446CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR (2012) Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149(7):1635–1646CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Fu Y, Dominissini D, Rechavi G, He C (2014) Gene expression regulation mediated through reversible m(6)A RNA methylation. Nat Rev Genet 15(5):293–306CrossRefPubMedGoogle Scholar
  14. 14.
    Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, Yi C, Lindahl T, Pan T, Yang YG, He C (2011) N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol 7(12):885–887CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Deng X, Chen K, Luo GZ, Weng X, Ji Q, Zhou T, He C (2015) Widespread occurrence of N6-methyladenosine in bacterial mRNA. Nucleic Acids Res 43:6557–6567CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Vitali P, Basyuk E, Le Meur E, Bertrand E, Muscatelli F, Cavaille J, Huttenhofer A (2005) ADAR2-mediated editing of RNA substrates in the nucleolus is inhibited by C/D small nucleolar RNAs. J Cell Biol 169(5):745–753CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Wang Y, Li Y, Toth JI, Petroski MD, Zhang Z, Zhao JC (2014) N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nat Cell Biol 16(2):191–198CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Wang X, Zhao BS, Roundtree IA, Lu Z, Han D, Ma H, Weng X, Chen K, Shi H, He C (2015) N(6)-methyladenosine modulates messenger RNA translation efficiency. Cell 161(6):1388–1399CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Liu N, Dai Q, Zheng G, He C, Parisien M, Pan T (2015) N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature 518(7540):560–564CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Kariko K, Muramatsu H, Keller JM, Weissman D (2012) Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin. Mol Ther 20(5):948–953CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Kariko K, Muramatsu H, Ludwig J, Weissman D (2011) Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res 39(21):e142CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Kariko K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, Weissman D (2008) Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther 16(11):1833–1840CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Heilman KL, Leach RA, Tuck MT (1996) Internal 6-methyladenine residues increase the in vitro translation efficiency of dihydrofolate reductase messenger RNA. Int J Biochem Cell Biol 28(7):823–829CrossRefPubMedGoogle Scholar
  24. 24.
    Simms CL, Hudson BH, Mosior JW, Rangwala AS, Zaher HS (2014) An active role for the ribosome in determining the fate of oxidized mRNA. Cell Rep 9(4):1256–1264CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Hudson BH, Zaher HS (2015) O6-Methylguanosine leads to position-dependent effects on ribosome speed and fidelity. RNA 21(9):1648–1659CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Hoernes TP, Clementi N, Faserl K, Glasner H, Breuker K, Lindner H, Hüttenhofer H, Erlacher MD (2016) Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code. Nucleic Acids Res 44(2):852–862CrossRefPubMedGoogle Scholar
  27. 27.
    Schägger H (2006) Tricine-SDS-PAGE. Nat Protoc 1(1):16–22 doi:nprot.2006.4CrossRefPubMedGoogle Scholar
  28. 28.
    Vazquez-Laslop N, Thum C, Mankin AS (2008) Molecular mechanism of drug-dependent ribosome stalling. Mol Cell 30(2):190–202CrossRefPubMedGoogle Scholar
  29. 29.
    Lang K, Micura R (2008) The preparation of site-specifically modified riboswitch domains as an example for enzymatic ligation of chemically synthesized RNA fragments. Nat Protoc 3(9):1457–1466CrossRefPubMedGoogle Scholar
  30. 30.
    Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T (2001) Cell-free translation reconstituted with purified components. Nat Biotechnol 19(8):751–755CrossRefPubMedGoogle Scholar
  31. 31.
    Erlacher MD, Chirkova A, Voegele P, Polacek N (2011) Generation of chemically engineered ribosomes for atomic mutagenesis studies on protein biosynthesis. Nat Protoc 6(5):580–592CrossRefPubMedGoogle Scholar
  32. 32.
    Schmid K, Thuring K, Keller P, Ochel A, Kellner S, Helm M (2015) Variable presence of 5-methylcytosine in commercial RNA and DNA. RNA Biol 12(10):1152–1158CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Bommer U, Burkhardt N, Jünemann R, Spahn CM, Triana-Alonso FJ, Nierhaus KH (1997) Ribosomes and polysomes. Subcellular fractionation—a practical approach. IRL Press, Washington, DC, pp 271–301Google Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Division of Genomics and RNomics, Biocenter InnsbruckMedical University of InnsbruckInnsbruckAustria

Personalised recommendations