Detection of endogenous LINE-1 ORF2p and its potent reverse transcriptase activity in the MCF-7 breast cancer cell...

Long interspersed element-1 (LINE-1 or L1) is actively jumping in humans, notably in germ cells, neurons, and certain types of cancer. An active L1 is ~6.0 kb in length and encodes two proteins, designated ORF1p and ORF2p. L1 RNA binds with L1-encoded proteins and forms L1-ribonucleoprotein particles (L1-RNPs), the retrotransposition intermediate. Although cells that support L1 retrotransposition express both proteins, the detection of ORF2 protein (ORF2p) is extremely challenging due to its limited expression and unavailability of a suitable antibody. Here, we characterize an anti-ORF2p antibody and show the presence of endogenous L1-ORF2p in multiple cancer cell lines, among which the MCF-7 cell line showed notably high expression. Complexes purified by immunoprecipitation (IP) with anti-ORF2p or anti-ORF1p from MCF-7 or HEK293T cells contain ORF2p and ORF1p and show ORF2p-mediated reverse transcriptase (RT) activity on L1, Alu, and GAPDH RNA templates. The ORF2 IP complex was further purified by size exclusion chromatography (SEC), which showed three major peaks with molecular weights around 796, 427, and 239 kDa. All three peaks showed the presence of L1 proteins, RNA, and ORF2p-mediated RT activity. Although many proteins have been identified that interact with L1 proteins, it is unclear which of these belong to the core L1 RNP. Our novel anti-ORF2p will provide a valuable resource for future studies involving ORF2p IP followed by SEC to identify the protein components of core L1 RNPs. In summary, we report the detection of endogenous L1 ORF2 protein and partial purification of its complex by ORF2p antibody-coupled IP and SEC.