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 language | English (US) |
---|---|
Title of host publication | Methods in Molecular Biology |
Publisher | Humana Press Inc. |
Pages | 133-146 |
Number of pages | 14 |
DOIs | |
State | Published - 2016 |
Publication series
Name | Methods in Molecular Biology |
---|---|
Volume | 1354 |
ISSN (Print) | 1064-3745 |
Bibliographical note
Funding Information:This work was supported by the National Institutes of Health’s National Cancer Institute RO1CA10888 and National Institutes of General Medical Sciences P30CA740300.
Publisher Copyright:
© Springer Science+Business Media New York 2016.
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