Skip to main content

Isolation of Cognate Cellular and Viral Ribonucleoprotein Complexes of HIV-1 RNA Applicable to Proteomic Discovery and Molecular Investigations

  • Protocol

Part of the Methods in Molecular Biology book series (MIMB,volume 1354)

Abstract

All decisions affecting the life cycle of human immunodeficiency virus (HIV-1) RNA are executed by ribonucleoprotein complexes (RNPs). HIV-1 RNA cycles through a progression of host RNPs composed of RNA-binding proteins regulating all stages of synthesis, processing, nuclear transport, translation, decay, and co-localization with assembling virions. RNA affinity chromatography is a versatile method to identify RNA-binding proteins to investigate the molecular basis of viral and cellular posttranscriptional control of gene expression. The bait is a HIV-1 RNA motif immobilized on a solid support, typically magnetic or Sepharose beads. The prey is pre-formed RNPs admixed in lysate from cells or concentrated virus particles. The methodology distinguishes high-affinity RNA-protein interactions from low-affinity complexes by increases in ionic strength during progressive elution cycles. Here, we describe RNA affinity chromatography of the 5′ untranslated region of HIV-1, obtaining mixtures of high-affinity RNA binding proteins suitable for mass spectrometry and proteome identification.

Key words

  • High-affinity RNA–protein interaction
  • Ribonucleoprotein particle (RNP)
  • Streptavidin-biotin affinity
  • Isotype-specific antibody
  • Immunoprecipitation
  • Magnetic beads
  • Cis-acting RNA element
  • Posttranscriptional control of gene expression

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

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-3046-3_9
  • Chapter length: 14 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   109.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-3046-3
  • 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   139.00
Price excludes VAT (USA)
Hardcover Book
USD   179.99
Price excludes VAT (USA)
Fig. 1

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

References

  1. Grüter P, Tabernero C, von Kobbe C, Schmitt C, Saavedra C, Bachi A, Wilm M, Felber BK, Izaurralde E (1998) TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus. Mol Cell 5:649–659

    CrossRef  Google Scholar 

  2. Hartman TR, Qian S, Bolinger C, Fernandez S, Schoenberg DR, Boris-Lawrie K (2006) RNA helicase A is necessary for translation of selected messenger RNAs. Nat Struct Mol Biol 13:509–516

    CAS  CrossRef  PubMed  Google Scholar 

  3. Tacheny A, Dieu M, Arnould T, Renard PJ (2013) Mass spectrometry-based identification of proteins interacting with nucleic acids. Proteomics 6:89–109

    CrossRef  Google Scholar 

  4. Stake M, Singh D, Singh G, Hernandez JM, Kaddis R, Parent LJ, Boris-Lawrie K (2015) HIV-1 and two avian retroviral 5’ untranslated regions bind orthologous human and chicken RNA binding proteins. Virology. NIHMS ID: 709312 (In Press)

    Google Scholar 

  5. Leblanc J, Weil J, Beemon K (2013) Posttranscriptional regulation of retroviral gene expression: primary RNA transcripts play three roles as pre-mRNA, mRNA, and genomic RNA. Wiley Interdiscip Rev RNA 4:567–580

    CAS  CrossRef  PubMed  Google Scholar 

  6. Butsch M, Boris-Lawrie K (2002) Destiny of unspliced retroviral RNA: ribosome and/or virion? J Virol 76:3089–3094

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  7. Wei P, Garber ME, Fang SM, Fischer WH, Jones KA (1998) A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92:451–462

    CAS  CrossRef  PubMed  Google Scholar 

  8. Bell NM, Kenyon JC, Balasubramanian S, Lever AM (2012) Comparative structural effects of HIV-1 Gag and nucleocapsid proteins in binding to and unwinding of the viral RNA packaging signal. Biochemistry 51:3162–3169

    CAS  CrossRef  PubMed  Google Scholar 

  9. Amarasinghe GK, Zhou J, Miskimon M, Chancellor KJ, McDonald JA, Matthews AG, Miller RR, Rouse MD, Summers MF (2001) Stem-loop SL4 of the HIV-1 psi RNA packaging signal exhibits weak affinity for the nucleocapsid protein, structural studies and implications for genome recognition. J Mol Biol 314:961–970

    CAS  CrossRef  PubMed  Google Scholar 

  10. Purcell DF, Martin MA (1993) Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity. J Virol 67:6365–6378

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Sharma A, Yilmaz A, Marsh K, Cochrane A, Boris-Lawrie K (2012) Thriving under stress: selective translation of HIV-1 structural protein mRNA during Vpr-mediated impairment of eIF4E translation activity. PLoS Pathog. doi:10.1371/journal.ppat.1002612

    Google Scholar 

Download references

Acknowledgements

This work was supported by the National Institutes of Health’s National Cancer Institute RO1CA10888 and National Institutes of General Medical Sciences P30CA740300.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Kathleen Boris-Lawrie .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Singh, D., Boeras, I., Singh, G., Boris-Lawrie, K. (2016). Isolation of Cognate Cellular and Viral Ribonucleoprotein Complexes of HIV-1 RNA Applicable to Proteomic Discovery and Molecular Investigations. In: Prasad, V., Kalpana, G. (eds) HIV Protocols. Methods in Molecular Biology, vol 1354. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3046-3_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3046-3_9

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3045-6

  • Online ISBN: 978-1-4939-3046-3

  • eBook Packages: Springer Protocols