Journal Article Ppublish
PubMed: 40568936 DOI: 10.1093/nar/gkaf526
Identification of a minimal Alu domain required for retrotransposition.
Nucleic Acids Res 53(12)
Authors: John B Moldovan, John Yin, John V Moran
Affiliations
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109, United States.
- Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, United States.
Abstract
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.
MeSH
Cells, Promoter Regions, Genetic, Nucleic Acids, Rna