A 3′ Poly(A) Tract Is Required for LINE-1 Retrotransposition

L1 retrotransposons express proteins (ORF1p and ORF2p) that preferentially mobilize their encoding RNA in cis, but they also can mobilize Alu RNA and, more rarely, cellular mRNAs in trans. Although these RNAs differ in sequence, each ends in a 3' polyadenosine (poly(A)) tract. Here, we replace the L1 polyadenylation signal with sequences derived from a non-polyadenylated long non-coding RNA (MALAT1), which can form a stabilizing triple helix at the 3' end of an RNA. L1/MALAT RNAs accumulate in cells, lack poly(A) tails, and are translated; however, they cannot retrotranspose in cis. Remarkably, the addition of a 16 or 40 base poly(A) tract downstream of the L1/MALAT triple helix restores retro-transposition in cis. The presence of a poly(A) tract also allows ORF2p to bind and mobilize RNAs in trans. Thus, a 3' poly(A) tract is critical for the retro-transposition of sequences that comprise approximately one billion base pairs of human DNA.