Skip to main content

Identification of RNA-Binding Proteins Associated to RNA Structural Elements

  • 785 Accesses

Part of the Methods in Molecular Biology book series (MIMB,volume 2323)

Abstract

RNA motifs guide the interaction with specific proteins leading to the assembly of ribonucleoprotein complexes that perform key functions in cellular processes. Internal ribosome entry site (IRES) elements are organized in structural domains that determine internal initiation of translation. In this chapter we describe a pull-down assay using streptavidin-aptamer tagged RNAs that combines RNA structure-dependent protein isolation with proteomic analysis to identify novel interactors recognizing RNA structural domains. This approach takes advantage of tRNA-scaffold guided expression, allowing the identification of factors belonging to networks involved in RNA and protein metabolism.

Key words

  • RNA structure
  • RNA-binding proteins
  • Pull-down
  • Streptavidin aptamer
  • tRNA scaffold
  • IRES elements
  • Translation control

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-0716-1499-0_9
  • Chapter length: 11 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   119.00
Price excludes VAT (USA)
  • ISBN: 978-1-0716-1499-0
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   159.99
Price excludes VAT (USA)
Hardcover Book
USD   219.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Hinnebusch AG (2017) Structural insights into the mechanism of scanning and start codon recognition in eukaryotic translation initiation. Trends Biochem Sci 42:589–611

    CAS  CrossRef  Google Scholar 

  2. Lozano G, Francisco-Velilla R, Martinez-Salas E (2018) Deconstructing internal ribosome entry site elements: an update of structural motifs and functional divergences. Open Biol 8(11):180155

    CAS  CrossRef  Google Scholar 

  3. Lozano G, Martinez-Salas E (2015) Structural insights into viral IRES-dependent translation mechanisms. Curr Opin Virol 12:113–120

    CAS  CrossRef  Google Scholar 

  4. Lozano G, Fernandez N, Martinez-Salas E (2016) Modeling three-dimensional structural motifs of viral IRES. J Mol Biol 428:767–776

    CAS  CrossRef  Google Scholar 

  5. Fernandez-Miragall O, Ramos R, Ramajo J, Martinez-Salas E (2006) Evidence of reciprocal tertiary interactions between conserved motifs involved in organizing RNA structure essential for internal initiation of translation. RNA 12:223–234

    CAS  CrossRef  Google Scholar 

  6. Jung S, Schlick T (2013) Candidate RNA structures for domain 3 of the foot-and-mouth-disease virus internal ribosome entry site. Nucleic Acids Res 41:1483–1495

    CAS  CrossRef  Google Scholar 

  7. Yu Y, Abaeva IS, Marintchev A, Pestova TV, Hellen CU (2011) Common conformational changes induced in type 2 picornavirus IRESs by cognate trans-acting factors. Nucleic Acids Res 39:4851–4865

    CAS  CrossRef  Google Scholar 

  8. Sweeney TR, Abaeva IS, Pestova TV, Hellen CU (2014) The mechanism of translation initiation on type 1 picornavirus IRESs. EMBO J 33:76–92

    CAS  CrossRef  Google Scholar 

  9. Pineiro D, Fernandez N, Ramajo J, Martinez-Salas E (2013) Gemin5 promotes IRES interaction and translation control through its C-terminal region. Nucleic Acids Res 41:1017–1028

    CAS  CrossRef  Google Scholar 

  10. Lee KM, Chen CJ, Shih SR (2017) Regulation mechanisms of viral IRES-driven translation. Trends Microbiol 25:546–561

    CAS  CrossRef  Google Scholar 

  11. Lozano G, Francisco-Velilla R, Martinez-Salas E (2018) Ribosome-dependent conformational flexibility changes and RNA dynamics of IRES domains revealed by differential SHAPE. Sci Rep 8:5545

    CrossRef  Google Scholar 

  12. Ponchon L, Beauvais G, Nonin-Lecomte S, Dardel F (2009) A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold. Nat Protoc 4:947–959

    CAS  CrossRef  Google Scholar 

  13. Fernandez-Chamorro J, Francisco-Velilla R, Ramajo J, Martinez-Salas E (2019) Rab1b and ARF5 are novel RNA-binding proteins involved in FMDV IRES-driven RNA localization. Life Sci Alliance 2(1):e201800131

    CrossRef  Google Scholar 

  14. Martinez-Salas E, Francisco-Velilla R, Fernandez-Chamorro J, Lozano G, Diaz-Toledano R (2015) Picornavirus IRES elements: RNA structure and host protein interactions. Virus Res 206:62–73

    CAS  CrossRef  Google Scholar 

  15. Mi H, Huang X, Muruganujan A, Tang H, Mills C, Kang D, Thomas PD (2017) PANTHER version 11: expanded annotation data from gene ontology and Reactome pathways, and data analysis tool enhancements. Nucleic Acids Res 45:D183–D189

    CAS  CrossRef  Google Scholar 

  16. Francisco-Velilla R, Fernandez-Chamorro J, Lozano G, Diaz-Toledano R, Martinez-Salas E (2015) RNA-protein interaction methods to study viral IRES elements. Methods 91:3–12

    CAS  CrossRef  Google Scholar 

  17. Lopez de Quinto S, Martinez-Salas E (2000) Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo. RNA 6:1380–1392

    CAS  CrossRef  Google Scholar 

  18. Francisco-Velilla R, Fernandez-Chamorro J, Ramajo J, Martinez-Salas E (2016) The RNA-binding protein Gemin5 binds directly to the ribosome and regulates global translation. Nucleic Acids Res 44:8335–8351

    CrossRef  Google Scholar 

Download references

Acknowledgments

We thank L. Ponchon for reagents, J. Ramajo for technical assistance, and A. Escos for comments on the protocol. This work was supported by MINECO (grant BFU2017-84492-R), Comunidad de Madrid (B2017/BMD-3770) and an Institutional grant from Fundación Ramón Areces.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Encarnacion Martinez-Salas .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Fernandez-Chamorro, J., Francisco-Velilla, R., Embarc-Buh, A., Martinez-Salas, E. (2021). Identification of RNA-Binding Proteins Associated to RNA Structural Elements. In: Ponchon, L. (eds) RNA Scaffolds. Methods in Molecular Biology, vol 2323. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1499-0_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1499-0_9

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1498-3

  • Online ISBN: 978-1-0716-1499-0

  • eBook Packages: Springer Protocols