Characterization of LINE-1 Ribonucleoprotein Particles.

A. Doucet (1, 2) , A. Hulme (2) , E. Sahinovic (1) , D. Kulpa (2) , J. Moldovan (2) , H. Kopera (2) , J. Athanikar (2) , M. Hasnaoui (1) , A. Bucheton (1) , J. Morvan (2, 3) , N. Gilbert (1)

1 IGH - Institut de génétique humaine
2 University of Michigan Medical School [Ann Arbor]
3 HHMI - Howard Hughes Medical Institute [Chevy Chase]

The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility (i.e., retrotransposition). Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1-encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition.

Domaines: Génétique humaine

A. Doucet, A. Hulme, E. Sahinovic, D. Kulpa, J. Moldovan, et al.. Characterization of LINE-1 Ribonucleoprotein Particles.. PLoS Genetics, 2010, 6 (10), pp.e1001150. ⟨10.1371/journal.pgen.1001150⟩. ⟨hal-00527796⟩