Sequence‐specific single‐strand RNA binding protein encoded by the human LINE‐1 retrotransposon

Previous experiments using human teratocarcinoma cells indicated that p40, the protein encoded by the first open reading frame (ORF) of the human LINE‐1 (L1Hs) retrotransposon, occurs in a large cytoplasmic ribonucleoprotein complex in direct association with L1Hs RNA(s), the p40 RNP complex. We have now investigated the interaction between partially purified p40 and L1Hs RNA in vitro using an RNA binding assay dependent on co‐immunoprecipitation of p40 and bound RNA. These experiments identified two p40 binding sites on the full‐length sense strand L1Hs RNA. Both sites are in the second ORF of the 6000 nt RNA: site A between residues 1999 and 2039 and site B between residues 4839 and 4875. The two RNA segments share homologous regions. Experiments involving UV cross‐linking followed by immunoprecipitation indicate that p40 in the in vitro complex is directly associated with L1Hs RNA, as it is in the p40 RNP complex found in teratocarcinoma cells. Binding and competition experiments demonstrate that p40 binds to single‐stranded RNA containing a p40 binding site, but not to single‐stranded or double‐stranded DNA, double‐stranded RNA or a DNA–RNA hybrid containing a binding site sequence. Thus, p40 appears to be a sequence‐specific, single‐strand RNA binding protein.