Characterization of LINE-1 ribonucleoprotein particles.
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.
Doucet
Aurélien J
AJ
Institut de Génétique Humaine, CNRS, UPR 1142, Montpellier, France. adoucet@umich.edu
Hulme
Amy E
AE
Sahinovic
Elodie
E
Kulpa
Deanna A
DA
Moldovan
John B
JB
Kopera
Huira C
HC
Athanikar
Jyoti N
JN
Hasnaoui
Manel
M
Bucheton
Alain
A
Moran
John V
JV
Gilbert
Nicolas
N
D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D015870:Gene Expression; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics*; D016254:Mutagenesis, Insertional; D009154:Mutation; D016366:Open Reading Frames / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D012261:Ribonucleoproteins; D012313:RNA; D012194:RNA-Directed DNA Polymerase