title: Isolation of Cognate Cellular and Viral Ribonucleoprotein Complexes of HIV-1 RNA Applicable to Proteomic Discovery and Molecular Investigations.
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.
authors.affiliation: Department of Veterinary Biosciences, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.|Center for Retrovirus Research, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.|Center for RNA Biology, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.
authors.forename: Deepali
authors.initials: D
authors.lastname: Singh
authors.affiliation: Department of Veterinary Biosciences, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.|Center for Retrovirus Research, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.|Center for RNA Biology, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.
authors.forename: Ioana
authors.initials: I
authors.lastname: Boeras
authors.affiliation: Department of Veterinary Biosciences, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.|Center for Retrovirus Research, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.|Center for RNA Biology, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.
authors.forename: Gatikrushna
authors.initials: G
authors.lastname: Singh
authors.affiliation: Department of Veterinary Biosciences, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA. boris-lawrie.1@osu.edu.|Center for Retrovirus Research, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA. boris-lawrie.1@osu.edu.|Center for RNA Biology, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA. boris-lawrie.1@osu.edu.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Boris-Lawrie
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D005786:Gene Expression Regulation; D015658:HIV Infections / Q000378:metabolism* / Q000821:virology*; D015497:HIV-1 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008214:Lymphocytes / Q000378:metabolism / Q000821:virology; D040901:Proteomics / Q000379:methods; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D014764:Viral Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D012367:RNA, Viral; D012261:Ribonucleoproteins; D014764:Viral Proteins
keywords: Cis-acting RNA element; High-affinity RNA–protein interaction; Immunoprecipitation; Isotype-specific antibody; Magnetic beads; Posttranscriptional control of gene expression; Ribonucleoprotein particle (RNP); Streptavidin-biotin affinity