title: Characterization of L1-Ribonucleoprotein Particles.
abstract: The LINE-1 retrotransposon (L1) encodes two proteins, ORF1p and ORF2p, which bind to the L1 RNA in cis, forming a ribonucleoprotein (RNP) complex that is critical for retrotransposition. Interactions with both permissive and repressive host factors pervade every step of the L1 life cycle. Until recently, limitations in detection and production precluded in-depth characterization of L1 RNPs. Inducible expression and recombinant engineering of epitope tags have made detection of both L1 ORFs routine. Here, we describe large-scale production of L1-expressing HEK-293T cells in suspension cell culture, cryomilling and affinity capture of L1 RNP complexes, sample preparation for analysis by mass spectrometry, and assay using the L1 element amplification protocol (LEAP) and qRT-PCR.
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.|Department of Pathology, Massachusetts General Hospital, Baltimore, MA, 21205, USA.
authors.forename: Martin S
authors.initials: MS
authors.lastname: Taylor
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, 10021, USA.|Department of Biochemistry and Molecular Pharmacology and Institute for Systems Genetics, New York University Langone School of Medicine, 550 First Avenue, New York, NY, 10016, USA.
authors.forename: John
authors.initials: J
authors.lastname: LaCava
authors.affiliation: Department of Molecular Biology and Genetics, High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.|Modern Meadow, Inc., Brooklyn, NY, 11220, USA.
authors.forename: Lixin
authors.initials: L
authors.lastname: Dai
authors.affiliation: Department of Biochemistry and Molecular Pharmacology and Institute for Systems Genetics, New York University Langone School of Medicine, 550 First Avenue, New York, NY, 10016, USA.
authors.forename: Paolo
authors.initials: P
authors.lastname: Mita
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
authors.forename: Kathleen H
authors.initials: KH
authors.lastname: Burns
authors.affiliation: Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, 10021, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Rout
authors.affiliation: Department of Biochemistry and Molecular Pharmacology and Institute for Systems Genetics, New York University Langone School of Medicine, 550 First Avenue, New York, NY, 10016, USA. Jef.Boeke@nyumc.org.
authors.forename: Jef D
authors.initials: JD
authors.lastname: Boeke
mesh_terms: D018929:Cell Culture Techniques; D002846:Chromatography, Affinity / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D020084:Long Interspersed Nucleotide Elements; D013058:Mass Spectrometry; D046912:Multiprotein Complexes / Q000302:isolation & purification / Q000378:metabolism; D016366:Open Reading Frames; D011485:Protein Binding; D060888:Real-Time Polymerase Chain Reaction; D012261:Ribonucleoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D012261:Ribonucleoproteins
keywords: Affinity purification; Cryomilling; Interactomics; LINE-1; Mass spectrometry; Metabolic labeling; Protein complexes; Ribonucleoprotein