Characterization of L1-ribonucleoprotein particles

Martin S. Taylor, John La Cava, Lixin Dai, Paolo Mita, Kathleen H. Burns, Michael P. Rout, Jef D. Boeke

    Research output: Chapter in Book/Report/Conference proceedingChapter

    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.

    Original languageEnglish (US)
    Title of host publicationMethods in Molecular Biology
    PublisherHumana Press Inc.
    Pages311-338
    Number of pages28
    DOIs
    StatePublished - 2016

    Publication series

    NameMethods in Molecular Biology
    Volume1400
    ISSN (Print)1064-3745

    Keywords

    • Affinity purification
    • Cryomilling
    • Interactomics
    • LINE-1
    • Mass spectrometry
    • Metabolic labelling
    • Protein complexes
    • Ribonucleoprotein

    ASJC Scopus subject areas

    • Molecular Biology
    • Genetics

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  • Cite this

    Taylor, M. S., La Cava, J., Dai, L., Mita, P., Burns, K. H., Rout, M. P., & Boeke, J. D. (2016). Characterization of L1-ribonucleoprotein particles. In Methods in Molecular Biology (pp. 311-338). (Methods in Molecular Biology; Vol. 1400). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-3372-3_20