Isolation of cognate cellular and viral ribonucleoprotein complexes of HIV-1 rna applicable to proteomic discovery and molecular investigations

Deepali Singh, Ioana Boeras, Gatikrushna Singh, Kathleen Boris-Lawrie

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages133-146
Number of pages14
DOIs
StatePublished - Jan 1 2016

Publication series

NameMethods in Molecular Biology
Volume1354
ISSN (Print)1064-3745

Keywords

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

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