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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References
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
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
Tacheny A, Dieu M, Arnould T, Renard PJ (2013) Mass spectrometry-based identification of proteins interacting with nucleic acids. Proteomics 6:89–109
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)
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
Butsch M, Boris-Lawrie K (2002) Destiny of unspliced retroviral RNA: ribosome and/or virion? J Virol 76:3089–3094
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
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
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
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
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
Acknowledgements
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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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
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DOI: https://doi.org/10.1007/978-1-4939-3046-3_9
Publisher Name: Humana Press, New York, NY
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