Chapter title |
Isolation of Cognate Cellular and Viral Ribonucleoprotein Complexes of HIV-1 RNA Applicable to Proteomic Discovery and Molecular Investigations
|
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Chapter number | 9 |
Book title |
HIV Protocols
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Published in |
Methods in molecular biology, January 2016
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DOI | 10.1007/978-1-4939-3046-3_9 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3045-6, 978-1-4939-3046-3
|
Authors |
Deepali Singh, Ioana Boeras, Gatikrushna Singh, Kathleen Boris-Lawrie, Singh, Deepali, Boeras, Ioana, Singh, Gatikrushna, Boris-Lawrie, Kathleen |
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. |
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