LINEs mobilize their own copies via retrotransposition. LINEs can be divided into two types. One is a stringent type, which constitutes a majority of LINEs. The other is a relaxed type. To elucidate the molecular mechanism of retrotransposition, we used here two different zebrafish LINEs belonging to the stringent type. By using retrotransposition assays, we demonstrated that proteins (ORF2) encoded by an individual LINE recognize the cognate 3' tail sequence of the LINE RNA strictly. By conducting in vitro binding assays with a variety of ORF2 proteins, we demonstrated that the region between the endonuclease and reverse transcriptase domains in ORF2 is the site at which the proteins bind the stem-loop structure of the 3' tail RNA, showing that the strict recognition of the stem-loop structure by the cognate ORF2 protein is an important step in retrotransposition. This recognition can be bipartite, involving the general recognition of the stem by cTBR (conserved tail-binding region) of ORF2 and the specific recognition of the loop by vTBR (variable tail-binding region). This is the first report that clearly characterized the RNA-binding region in ORF2, providing the generality for the recognition mechanism of the RNA tail by the ORF2 protein encoded by LINEs.
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