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Characterization of L1-Ribonucleoprotein Particles

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Transposons and Retrotransposons

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1400))

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.

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Acknowledgements

We thank Dan Leahy, Jennifer Kavran, and Yana Li for help with suspension cell culture. This work was supported in part by NIH grant U54GM103511 to MPR, grant 5P50GM107632 to JDB, KHB, and MPR, U54GM103520 to JDB, R01CA163705 and R01GM103999 to KHB, and US DoD grant OC120390 to KHB.

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Authors and Affiliations

  1. Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Martin S. Taylor & Kathleen H. Burns

  2. Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, 10021, USA

    John LaCava & Michael P. Rout

  3. 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

    John LaCava, Paolo Mita & Jef D. Boeke Ph.D.

  4. Department of Molecular Biology and Genetics, High Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Lixin Dai

  5. Modern Meadow, Inc., Brooklyn, NY, 11220, USA

    Lixin Dai

  6. Department of Pathology, Massachusetts General Hospital, Baltimore, MA, 21205, USA

    Martin S. Taylor

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  1. Martin S. Taylor
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  2. John LaCava
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  3. Lixin Dai
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  4. Paolo Mita
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  5. Kathleen H. Burns
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  7. Jef D. Boeke Ph.D.
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Corresponding author

Correspondence to Jef D. Boeke Ph.D. .

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Editors and Affiliations

  1. Institute of Genetics and Molecular Medi, Genyo (Center for Genomics and Oncologic, Granada, Spain

    Jose L. Garcia-Pérez

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Taylor, M.S. et al. (2016). Characterization of L1-Ribonucleoprotein Particles. In: Garcia-Pérez, J. (eds) Transposons and Retrotransposons. Methods in Molecular Biology, vol 1400. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3372-3_20

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  • DOI: https://doi.org/10.1007/978-1-4939-3372-3_20

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3370-9

  • Online ISBN: 978-1-4939-3372-3

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