title: Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution.
abstract: Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes. The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.
authors.affiliation: Department of Veterinary & Biomedical Sciences, University of Minnesota.
authors.forename: Gatikrushna
authors.initials: G
authors.lastname: Singh
authors.affiliation: Department of Veterinary Biosciences, Ohio State University.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Fritz
authors.affiliation: Department of Veterinary Biosciences, Ohio State University.
authors.forename: Arnaz
authors.initials: A
authors.lastname: Ranji
authors.affiliation: School of Biotechnology, Gautam Buddha University.
authors.forename: Deepali
authors.initials: D
authors.lastname: Singh
authors.affiliation: Department of Veterinary & Biomedical Sciences, University of Minnesota; Department of Veterinary Biosciences, Ohio State University; kbl@umn.edu.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Boris-Lawrie
mesh_terms: D005786:Gene Expression Regulation; D006801:Humans; D009841:Oligonucleotides; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics; D012261:Ribonucleoproteins / Q000302:isolation & purification*
chemical_list: D009841:Oligonucleotides; D012333:RNA, Messenger; D012261:Ribonucleoproteins