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 ri...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2010-10-01
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| Series: | PLoS Genetics |
| Online Access: | http://europepmc.org/articles/PMC2951350?pdf=render |
| _version_ | 1828531472583098368 |
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| author | Aurélien J Doucet Amy E Hulme Elodie Sahinovic Deanna A Kulpa John B Moldovan Huira C Kopera Jyoti N Athanikar Manel Hasnaoui Alain Bucheton John V Moran Nicolas Gilbert |
| author_facet | Aurélien J Doucet Amy E Hulme Elodie Sahinovic Deanna A Kulpa John B Moldovan Huira C Kopera Jyoti N Athanikar Manel Hasnaoui Alain Bucheton John V Moran Nicolas Gilbert |
| author_sort | Aurélien J Doucet |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-11T22:39:06Z |
| format | Article |
| id | doaj.art-f1a91967e47a4907be4db4b3c6eb6921 |
| institution | Directory Open Access Journal |
| issn | 1553-7390 1553-7404 |
| language | English |
| last_indexed | 2024-12-11T22:39:06Z |
| publishDate | 2010-10-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Genetics |
| spelling | doaj.art-f1a91967e47a4907be4db4b3c6eb69212022-12-22T00:47:52ZengPublic Library of Science (PLoS)PLoS Genetics1553-73901553-74042010-10-0161010.1371/journal.pgen.1001150Characterization of LINE-1 ribonucleoprotein particles.Aurélien J DoucetAmy E HulmeElodie SahinovicDeanna A KulpaJohn B MoldovanHuira C KoperaJyoti N AthanikarManel HasnaouiAlain BuchetonJohn V MoranNicolas GilbertThe 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.http://europepmc.org/articles/PMC2951350?pdf=render |
| spellingShingle | Aurélien J Doucet Amy E Hulme Elodie Sahinovic Deanna A Kulpa John B Moldovan Huira C Kopera Jyoti N Athanikar Manel Hasnaoui Alain Bucheton John V Moran Nicolas Gilbert Characterization of LINE-1 ribonucleoprotein particles. PLoS Genetics |
| title | Characterization of LINE-1 ribonucleoprotein particles. |
| title_full | Characterization of LINE-1 ribonucleoprotein particles. |
| title_fullStr | Characterization of LINE-1 ribonucleoprotein particles. |
| title_full_unstemmed | Characterization of LINE-1 ribonucleoprotein particles. |
| title_short | Characterization of LINE-1 ribonucleoprotein particles. |
| title_sort | characterization of line 1 ribonucleoprotein particles |
| url | http://europepmc.org/articles/PMC2951350?pdf=render |
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