title: A versatile tandem RNA isolation procedure to capture in vivo formed mRNA-protein complexes.
abstract: We describe a tandem RNA isolation procedure (TRIP) that enables purification of in vivo formed messenger ribonucleoprotein (mRNP) complexes. The procedure relies on the purification of polyadenylated mRNAs with oligo(dT) beads from cellular extracts, followed by the capture of specific mRNAs with 3'-biotinylated 2'-O-methylated antisense RNA oligonucleotides, which are recovered with streptavidin beads. TRIP was applied to isolate in vivo crosslinked mRNP complexes from yeast, nematodes and human cells for subsequent analysis of RNAs and bound proteins. The method provides a basis for adaptation to other types of polyadenylated RNAs, enabling the comprehensive identification of bound proteins/RNAs, and the investigation of dynamic rearrangement of mRNPs imposed by cellular or environmental cues.
authors.affiliation: Dept. of Microbial and Cellular Sciences, School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH, United Kingdom.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Matia-González
authors.affiliation: Dept. of Microbial and Cellular Sciences, School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH, United Kingdom.
authors.forename: Valentina
authors.initials: V
authors.lastname: Iadevaia
authors.affiliation: Dept. of Microbial and Cellular Sciences, School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH, United Kingdom. Electronic address: a.gerber@surrey.ac.uk.
authors.forename: André P
authors.initials: AP
authors.lastname: Gerber
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019921:Biotinylation; D015153:Blotting, Western / Q000379:methods; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008863:Microspheres; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020320:Oligoribonucleotides, Antisense / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry; D014466:Ultraviolet Rays
chemical_list: D009838:Oligodeoxyribonucleotides; D020320:Oligoribonucleotides, Antisense; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; C026452:messenger ribonucleoprotein; C027903:oligo (dT); D019809:Streptavidin
keywords: Antisense oligonucleotides; Non-coding RNA; Post-transcriptional regulation; RNA-binding protein; RNA-protein interaction; Ribonucleoprotein complex