Details

Autor(en) / Beteiligte

• Doucet, Aurélien J
• Hulme, Amy E
• Sahinovic, Elodie
• Kulpa, Deanna A
• Moldovan, John B
• Kopera, Huira C
• Athanikar, Jyoti N
• Hasnaoui, Manel
• Bucheton, Alain
• Moran, John V
• Gilbert, Nicolas
• Barsh, Gregory S.

Titel

Characterization of LINE-1 ribonucleoprotein particles

Ist Teil von

• PLoS genetics, 2010-10, Vol.6 (10), p.e1001150

Ort / Verlag

United States: Public Library of Science

Erscheinungsjahr

2010

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.