Expression and detection of LINE-1 ORF-encoded proteins

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.

The Flow of the Gibbon LAVA Element Is Facilitated by the LINE-1 Retrotransposition Machinery

“…In the case of the anti-L1 ORF1p immunoblot analysis (left panel), 20 µg of the cell lysate isolated from each of the differently transfected HeLa-HA cell cultures, were loaded per lane on a 12% PAA gel. Because ORF2p (Predicted MW∼150 kDa) is expressed at a significantly lower level than ORF1p (Dai et al 2014), 40 µg of total cell lysate from each of the differently transfected HeLa-HA cells were loaded per lane on a 6% PAA gel to perform anti-L1 ORF2p immunoblot analysis (right panel). Detectable amounts of intact L1 ORF1-encoded proteins are absent from pJM101/L1 RP ΔneoΔORF1-transfected HeLa cells.…”

Ductal cells of minor salivary glands in Sjögren's syndrome express LINE‐1 ORF2p and APOBEC3B

“…L1 ORF2p detection was hindered until recently, due to the high percentage of L1 adenosine content (up to 40%) that may cause a defective transcription elongation, or the unconventional translation mechanism of L1 RNA that results in low levels of L1 ORF2p (~one ORF2p molecule per L1 mRNA) . The antibody applied in the present study recognizes the C‐terminal region of ORF2 that remains in the cytoplasm after truncation of the C‐terminal fragment, a process that is necessary for the nuclear entrance of L1 ORF2p .…”

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

“…Nevertheless, new inserts created through transposition can generate genetic diversity but can also cause disease . The L1 element encodes two open reading frame proteins, ORF1p and ORF2p, that interact with the L1 transcript to form a ribonucleoprotein (RNP) complex that is required for transposing its own RNA copy back into the host genome . We have yet to fully understand the detailed mechanisms by which L1 RNPs and host cellular proteins co‐exist.…”