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

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


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

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

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Correspondence to Kathleen Boris-Lawrie .

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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.

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3045-6

  • Online ISBN: 978-1-4939-3046-3

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