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 - Jan 1 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

Fingerprint Dive into the research topics of 'Characterization of L1-ribonucleoprotein particles'. Together they form a unique fingerprint.

  • 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