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Mob Genet Elements. 2014 May 22;4:e29319. eCollection 2014.

Expression and detection of LINE-1 ORF-encoded proteins.

Author information

1
High Throughput Biology Center and Department of Molecular Biology and Genetics; Johns Hopkins University School of Medicine; Baltimore, MD USA.
2
Laboratory of Cellular and Structural Biology; The Rockefeller University; New York, NY USA.
3
High Throughput Biology Center and Department of Pharmacology and Molecular Sciences; Johns Hopkins University School of Medicine; Baltimore, MD USA.
4
Institute for Systems Genetics; New York University School of Medicine; New York, NY USA.

Abstract

LINE-1 (L1) elements are endogenous retrotransposons active in mammalian genomes. The L1 RNA is bicistronic, encoding two non-overlapping open reading frames, ORF1 and ORF2, whose protein products (ORF1p and ORF2p) bind the L1 RNA to form a ribonucleoprotein (RNP) complex that is presumed to be a critical retrotransposition intermediate. However, ORF2p is expressed at a significantly lower level than ORF1p; these differences are thought to be controlled at the level of translation, due to a low frequency ribosome reinitiation mechanism controlling ORF2 expression. As a result, while ORF1p is readily detectable, ORF2p has previously been very challenging to detect in vitro and in vivo. To address this, we recently tested several epitope tags fused to the N- or C-termini of the ORF proteins in an effort to enable robust detection and affinity purification from native (L1RP) and synthetic (ORFeus-Hs) L1 constructs. An analysis of tagged RNPs from both L1RP and ORFeus-Hs showed similar host-cell-derived protein interactors. Our observations also revealed that the tag sequences affected the retrotransposition competency of native and synthetic L1s differently although they encode identical ORF proteins. Unexpectedly, we observed apparently stochastic expression of ORF2p within seemingly homogenous L1-expressing cell populations.

KEYWORDS:

I-DIRT; LINE-1; epitope tag; mass spectrometry; synthetic transposon

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