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RNA Chaperones pp 179-192 | Cite as

RNA Remodeling by RNA Chaperones Monitored by RNA Structure Probing

  • Susann FriedrichEmail author
  • Tobias Schmidt
  • Sven-Erik BehrensEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2106)

Abstract

RNA structure probing enables the characterization of RNA secondary structures by established procedures such as the enzyme- or chemical-based detection of single- or double-stranded regions. A specific type of application involves the detection of changes of RNA structures and conformations that are induced by proteins with RNA chaperone activity. This chapter outlines a protocol to analyze RNA structures in vitro in the presence of an RNA-binding protein with RNA chaperone activity. For this purpose, we make use of the methylating agents dimethyl sulfate (DMS) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMCT). DMS and CMCT specifically modify nucleotides that are not involved in base-pairing or tertiary structure hydrogen bonding and that are not protected by a ligand such as a protein. Modified bases are identified by primer extension. As an example, we describe how the RNA chaperone activity of an isoform of the RNA-binding protein AUF1 induces the flaviviral RNA switch required for viral genome cyclization and viral replication.

This chapter includes comprehensive protocols for in vitro synthesis of RNA, 32P-5′-end labeling of DNA primers, primer extension, as well as the preparation and running of analytical gels. The described methodology should be applicable to any other RNA and protein of interest to identify protein-directed RNA remodeling.

Key words

RNA structure RNA chaperone RNA remodeling DMS CMCT 

Notes

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (grants BE1885/7-1/2 and BE1885/12-1 to S.F. and S.-E.B.).

References

  1. 1.
    Rajkowitsch L, Chen D, Stampfl S, Semrad K, Waldsich C, Mayer O, Jantsch MF, Konrat R, Blasi U, Schroeder R (2007) RNA chaperones, RNA annealers and RNA helicases. RNA Biol 4:118–130CrossRefGoogle Scholar
  2. 2.
    White EJ, Matsangos AE, Wilson GM (2017) AUF1 regulation of coding and noncoding RNA. Wiley Interdiscip Rev RNA 8:e1393CrossRefGoogle Scholar
  3. 3.
    Friedrich S, Schmidt T, Geissler R, Lilie H, Chabierski S, Ulbert S, Liebert UG, Golbik RP, Behrens SE (2014) AUF1 p45 promotes West Nile virus replication by an RNA chaperone activity that supports cyclization of the viral genome. J Virol 88:11586–11599CrossRefGoogle Scholar
  4. 4.
    Friedrich S, Schmidt T, Schierhorn A, Lilie H, Szczepankiewicz G, Bergs S, Liebert UG, Golbik RP, Behrens SE (2016) Arginine methylation enhances the RNA chaperone activity of the West Nile virus host factor AUF1 p45. RNA 22:1574–1591CrossRefGoogle Scholar
  5. 5.
    Friedrich S, Engelmann S, Schmidt T, Szczepankiewicz G, Bergs S, Liebert UG, Kummerer BM, Golbik RP, Behrens SE (2018) The host factor AUF1 p45 supports flavivirus propagation by triggering the RNA switch required for viral genome cyclization. J Virol 92:JVI.01647-17CrossRefGoogle Scholar
  6. 6.
    Ehresmann C, Baudin F, Mougel M, Romby P, Ebel JP, Ehresmann B (1987) Probing the structure of RNAs in solution. Nucleic Acids Res 15:9109–9128CrossRefGoogle Scholar
  7. 7.
    Brunel C, Romby P (2000) Probing RNA structure and RNA-ligand complexes with chemical probes. Methods Enzymol 318:3–21CrossRefGoogle Scholar
  8. 8.
    Regulski EE, Breaker RR (2008) In-line probing analysis of riboswitches. Methods Mol Biol 419:53–67CrossRefGoogle Scholar
  9. 9.
    Wakeman CA, Winkler WC (2009) Analysis of the RNA backbone: structural analysis of riboswitches by in-line probing and selective 2′-hydroxyl acylation and primer extension. Methods Mol Biol 540:173–191CrossRefGoogle Scholar
  10. 10.
    Wilkinson KA, Merino EJ, Weeks KM (2006) Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nat Protoc 1:1610–1616CrossRefGoogle Scholar
  11. 11.
    Mortimer SA, Weeks KM (2007) A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry. J Am Chem Soc 129:4144–4145CrossRefGoogle Scholar
  12. 12.
    Mortimer SA, Weeks KM (2009) Time-resolved RNA SHAPE chemistry: quantitative RNA structure analysis in one-second snapshots and at single-nucleotide resolution. Nat Protoc 4:1413–1421CrossRefGoogle Scholar
  13. 13.
    Senecoff JF, Meagher RB (1992) In vivo analysis of plant RNA structure: soybean 18S ribosomal and ribulose-1,5-bisphosphate carboxylase small subunit RNAs. Plant Mol Biol 18:219–234CrossRefGoogle Scholar
  14. 14.
    Zaug AJ, Cech TR (1995) Analysis of the structure of Tetrahymena nuclear RNAs in vivo: telomerase RNA, the self-splicing rRNA intron, and U2 snRNA. RNA 1:363–374PubMedPubMedCentralGoogle Scholar
  15. 15.
    Wells SE, Hughes JM, Igel AH, Ares M Jr (2000) Use of dimethyl sulfate to probe RNA structure in vivo. Methods Enzymol 318:479–493CrossRefGoogle Scholar
  16. 16.
    Waldsich C, Grossberger R, Schroeder R (2002) RNA chaperone StpA loosens interactions of the tertiary structure in the td group I intron in vivo. Genes Dev 16:2300–2312CrossRefGoogle Scholar
  17. 17.
    Zemora G, Waldsich C (2010) RNA folding in living cells. RNA Biol 7:634–641CrossRefGoogle Scholar
  18. 18.
    Andrade JM, Dos Santos RF, Chelysheva I, Ignatova Z, Arraiano CM (2018) The RNA-binding protein Hfq is important for ribosome biogenesis and affects translation fidelity. EMBO J 37:e97631CrossRefGoogle Scholar
  19. 19.
    Moazed D, Robertson JM, Noller HF (1988) Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA. Nature 334:362–364CrossRefGoogle Scholar
  20. 20.
    Mohr S, Stryker JM, Lambowitz AM (2002) A DEAD-box protein functions as an ATP-dependent RNA chaperone in group I intron splicing. Cell 109:769–779CrossRefGoogle Scholar
  21. 21.
    Chakshusmathi G, Kim SD, Rubinson DA, Wolin SL (2003) A La protein requirement for efficient pre-tRNA folding. EMBO J 22:6562–6572CrossRefGoogle Scholar
  22. 22.
    Ross JA, Ellis MJ, Hossain S, Haniford DB (2013) Hfq restructures RNA-IN and RNA-OUT and facilitates antisense pairing in the Tn10/IS10 system. RNA 19:670–684CrossRefGoogle Scholar
  23. 23.
    Grohman JK, Gorelick RJ, Lickwar CR, Lieb JD, Bower BD, Znosko BM, Weeks KM (2013) A guanosine-centric mechanism for RNA chaperone function. Science 340:190–195CrossRefGoogle Scholar
  24. 24.
    Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Charles Tanford Protein Centre, Institute of Biochemistry and BiotechnologyMartin Luther University Halle-WittenbergHalleGermany

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