Characterization of L1-Ribonucleoprotein Particles

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.

Fig. 1

Suspension culture of HEK-293T_LD cells using square glass bottles. (a) Orbital shaker bottle setup in a CO₂-controlled humidified incubator cabinet. Total yield from 48 bottles was approximately 250 g wet cell weight (WCW), the equivalent of 2500 15 cm adherent culture plates. (b) Dr. Lixin Dai proudly showcases an incubator full of adherent culture plates for harvest. Harvesting this entire incubator full of cells yielded 16 g WCW, which can now be accomplished in three to four suspension bottles. (c) Diagram of modifications to Nalgene 4 × 10 configuration 20 mm test tube racks to fit square glass bottles. Dividers are cut with diagonal cutting pliers and smoothed with a file. Sections of rack can be removed with a bandsaw. The diversity of available rack sizes and shapes allows adaptation of this strategy for many size bottles

Fig. 2

Injection of pelleted human cells into liquid nitrogen to make “BB's.” After centrifugation in a capped syringe, cells are injected into 50 mL conical tubes containing liquid nitrogen. Injection at a moderate rate prevents over-clumping of the cells. Use a pre-chilled spatula to break up any clumps

Fig. 3

Cryomilling setup for human cells. (a) Cryomilling apparatus in the Resch PM 100. 125 mL grinding jar is shown with custom PTFE insulators above and below. (b) Comparison of simplified one-stage and prior two-stage grinding protocols on protein extraction and affinity capture reveals equivalent results. HEK-293T_LD cells expressing full-length L1 (ORFeus HS background) with ORF1p tagged with Flag (LD288) or GFP (LD258) were affinity captured (pullout) using respective epitope tag antibody-conjugated Dynabeads in our standard extraction solution and eluted under denaturing conditions. Total extracted lysate (lysate) before affinity capture is shown