Identification of a minimal <i>Alu</i> domain required for retrotransposition

Alu elements are primate-specific retrotransposons that comprise ∼11% of human DNA. Alu sequences contain an internal RNA polymerase III promoter, and the resultant Alu RNA transcripts mobilize by a replicative process termed retrotransposition, which requires the long interspersed element-1 open reading frame 2-encoded protein (ORF2p). Here, we used HeLa cell-based retrotransposition assays to define a minimal Alu domain necessary for retrotransposition. We demonstrate that Alu transcripts expressed from a cytomegalovirus (CMV) RNA polymerase II promoter can efficiently undergo retrotransposition. The use of an external CMV promoter to express Alu RNA allowed us to construct separation-of-function mutations to examine the effects of large deletions within the Alu sequence on retrotransposition. Deletion mutagenesis demonstrated that a 46-nucleotide (nt) domain located at the 5' end of the Alu RNA transcript is necessary for retrotransposition. Consistent with current models, the 46-nt 5' Alu domain associates with SRP9/14 in HeLa-HA cell extracts and can promote retrotransposition in HeLa-HA cells. We propose that the 46-nt 5' Alu domain forms a discrete structure that allows for SRP9/14 binding and ribosomal association, thereby allowing the Alu poly(A) tract to compete with the L1 poly(A) tail for ORF2p RNA binding to mediate its retrotransposition.