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Characterization of L1-Ribonucleoprotein Particles

  • Martin S. Taylor
  • John LaCava
  • Lixin Dai
  • Paolo Mita
  • Kathleen H. Burns
  • Michael P. Rout
  • Jef D. BoekeEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

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

Notes

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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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Martin S. Taylor
    • 1
    • 6
  • John LaCava
    • 2
    • 3
  • Lixin Dai
    • 4
    • 5
  • Paolo Mita
    • 3
  • Kathleen H. Burns
    • 1
  • Michael P. Rout
    • 2
  • Jef D. Boeke
    • 3
    Email author
  1. 1.Department of PathologyJohns Hopkins University School of MedicineBaltimoreUSA
  2. 2.Laboratory of Cellular and Structural BiologyThe Rockefeller UniversityNew YorkUSA
  3. 3.Department of Biochemistry and Molecular Pharmacology and Institute for Systems GeneticsNew York University Langone School of MedicineNew YorkUSA
  4. 4.Department of Molecular Biology and Genetics, High Throughput Biology CenterJohns Hopkins University School of MedicineBaltimoreUSA
  5. 5.Modern Meadow, Inc.BrooklynUSA
  6. 6.Department of PathologyMassachusetts General HospitalBaltimoreUSA

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