title: Rapid purification of ribosomal particles assembled on histone H4 mRNA: a new method based on mRNA-DNA chimaeras.
abstract: Detailed knowledge of the structure of the ribosomal particles during their assembly on mRNA is a prerequisite for understanding the intricate translation initiation process. In vitro preparation of eukaryotic translation initiation complexes is limited by the rather tricky assembly from individually purified ribosomal subunits, initiation factors and initiator tRNA. In order to directly isolate functional complexes from living cells, methods based on affinity tags have been developed which, however, often suffer from non-specific binding of proteins and/or RNAs. In the present study we present a novel method designed for the purification of high-quality ribosome/mRNA particles assembled in RRL (rabbit reticulocyte lysate). Chimaerical mRNA-DNA molecules, consisting of the full-length mRNA ligated to a biotinylated desoxy-oligonucleotide, are immobilized on streptavidin-coated beads and incubated with RRL to form initiation complexes. After a washing step, the complexes are eluted by specific DNase I digestion of the DNA moiety of the chimaera, releasing initiation complexes in native conditions. Using this simple and robust purification setup, 80S particles properly programmed with full-length histone H4 mRNA were isolated with the expected ribosome/mRNA molar ratio of close to 1. We show that by using this novel approach purified ribosomal particles can be obtained that are suitable for biochemical and structural studies, in particular single-particle cryo-EM (cryo-electron microscopy). This purification method thus is a versatile tool for the isolation of fully functional RNA-binding proteins and macromolecular RNPs.
authors.affiliation: Institut de Biologie Moléculaire et Cellulaire, 'Architecture et Réactivité de l'ARN' CNRS UPR9002, Université de Strasbourg, 15, rue René Descartes, 67084 Strasbourg, France.
authors.forename: Lydia
authors.initials: L
authors.lastname: Prongidi-Fix
authors.forename: Laure
authors.initials: L
authors.lastname: Schaeffer
authors.forename: Angelita
authors.initials: A
authors.lastname: Simonetti
authors.forename: Sharief
authors.initials: S
authors.lastname: Barends
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Ménétret
authors.forename: Bruno P
authors.initials: BP
authors.lastname: Klaholz
authors.forename: Gilbert
authors.initials: G
authors.lastname: Eriani
authors.forename: Franck
authors.initials: F
authors.lastname: Martin
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D020285:Cryoelectron Microscopy; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics*; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012261:Ribonucleoproteins / Q000302:isolation & purification*; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D004274:DNA, Recombinant; D006657:Histones; D012333:RNA, Messenger; D012261:Ribonucleoproteins