Proteome Profile of Endogenous Retrotransposon-Associated Complexes in Human Embryonic Stem Cells

Abstract

Long Interspersed Element-1 (LINE-1 or L1) are transposable elements similar to retroviruses that have existed in the genome of primates for millions of years. They encode two Open Reading Frame (ORF) proteins (ORF1p and ORF2p) that bind L1 RNA to form a ribonucleoprotein (RNP) complex and are required for L1 integration into the host genome. Humans have evolved with L1 and found ways to limit L1 activity. To identify partners of the L1 RNP, previous studies used ectopic expression of L1 ORF1/2p or RNA in various cancer cells, which express low levels of the ORF proteins. Whether naturally occurring L1 RNP interacts with the same proteins in non-cancer cells is unknown. Here, the aim is to examine the natural assembly of endogenous L1 RNPs in normal human cells. L1 elements are expressed in human embryonic stem cells (hESCs), derived from pre-implantation embryos. Therefore, these cells are used to immunoprecipitate ORF1p followed by MS to identify proteins that associate with the naturally-occurring L1 ORF1p. Some of the same proteins as well as unique proteins are found interacting with the endogenous L1 ORF1p complexes. The analysis of ORF1p-associated proteins in hESCs can help address important questions in both retrotransposon biology and the biology of hESCs.

Keywords: human embryonic stem cells; immunoprecipitation; long interspersed element-1; mass spectrometry; retrotransposition; ribonucleoprotein complex.

FIG 1.

ORF1p expression in hESCs. (A) IF imaging of ORF1p (left-green) and OCT4 (red) in hESCs, showing single staining of ORF1 (Left) and a merged image of staining for ORF1 (green), OCT4 (nuclei - red) and chromatin (nuclei - blue) (Right). Far right, negative controls for corresponding secondary antibodies. (B) IB analysis of cell extracts to verify expression of ORF1p in hESCs. Lanes 1 and 2 are input at 1 and 2 % from 1.2 mL extracts. The human embryonal carcinoma, NT2D, cell line is a positive control for ORF1p expression. M, molecular markers; ORF1p (40 kDa) band is below the 50 kDa mark. Right, IP of ORF1p and IgG. Asterisks indicate immunoglobulin heavy chain. (C) Table of mass spectrometry (MS) analysis showing p40 (ORF1) protein in three samples were immunoprecipitated with the anti-ORF1 (green) but not with the mouse IgG control (orange).

FIG 2.

Proteins identified with ORF1p in hESCs and functional annotation (A) Flowchart for IP. (B) Volcano plot of average abundance ratio (ORF1/IgG) for proteins with an abundance ratio and p-value that are quantifiable by the PD2.2 software. Abundance ratio and p-value are represented as log base 2 and 10, respectively. Green spots represent proteins with an average abundance ratio threshold of ≥2 and p-value of at least 0.010. (C) GO analysis of the 48 out of 65 proteins with annotated gene symbols. BP, Biological Process; CC, Cellular Component, MF, Molecular Function. (D) Networks of protein-protein interactions from 48 proteins. Only eight proteins are closely related. GO analysis of proteins shown in the network. Nodes are color coded as per the GO analysis.