Characterization of L1-Ribonucleoprotein Particles

Methods Mol Biol. 2016;1400:311-38. doi: 10.1007/978-1-4939-3372-3_20.

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.

Keywords: Affinity purification; Cryomilling; Interactomics; LINE-1; Mass spectrometry; Metabolic labeling; Protein complexes; Ribonucleoprotein.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cell Culture Techniques
  • Chromatography, Affinity / methods
  • HEK293 Cells
  • Humans
  • Long Interspersed Nucleotide Elements*
  • Mass Spectrometry
  • Multiprotein Complexes* / isolation & purification
  • Multiprotein Complexes* / metabolism
  • Open Reading Frames*
  • Protein Binding
  • Real-Time Polymerase Chain Reaction
  • Ribonucleoproteins / genetics*
  • Ribonucleoproteins / isolation & purification
  • Ribonucleoproteins / metabolism*

Substances

  • Multiprotein Complexes
  • Ribonucleoproteins