Identification of a minimal Alu domain required for retrotransposition

Alu elements are primate-specific retrotransposon sequences that comprise [~]11% of human genomic DNA. Alu sequences contain an internal RNA polymerase III promoter and the resultant Alu RNA transcripts mobilize by a replicative process termed retrotransposition. Alu retrotransposition requires the Long INterspersed Element-1 (LINE-1) open reading frame 2-encoded protein (ORF2p). Current models propose that Alu RNA binds to signal recognition particle proteins 9 and 14 (SRP9/14) and localizes to ribosomes, which allows Alu to hijack L1 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 Alu retrotransposition. Consistent with current models, the 46 nt 5' Alu domain associates with SRP9/14 in HeLa-HA cell extracts and can promote a single round of retrotransposition in HeLa-HA cells. We propose that the 46 nt 5' Alu domain forms a discrete structure that allows for SRP 9/14 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.