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Characterization of L1 ORF1p Self-Interaction and Cellular Localization Using a Mammalian Two-Hybrid System

Long INterspersed Element-1 (LINE-1, L1) is an active retrotransposon that mobilizes using a ribonucleoprotein particle (RNP) intermediate composed of the full-length bicistronic L1 mRNA and the two proteins (ORF1p and ORF2p) encoded by that mRNA. ORF1p and ORF2p demonstrate cis-preference for their...

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Autores principales: Sokolowski, Mark, deHaro, Dawn, Christian, Claiborne M., Kines, Kristine J., Belancio, Victoria P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3852968/
https://www.ncbi.nlm.nih.gov/pubmed/24324740
http://dx.doi.org/10.1371/journal.pone.0082021
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author Sokolowski, Mark
deHaro, Dawn
Christian, Claiborne M.
Kines, Kristine J.
Belancio, Victoria P.
author_facet Sokolowski, Mark
deHaro, Dawn
Christian, Claiborne M.
Kines, Kristine J.
Belancio, Victoria P.
author_sort Sokolowski, Mark
collection PubMed
description Long INterspersed Element-1 (LINE-1, L1) is an active retrotransposon that mobilizes using a ribonucleoprotein particle (RNP) intermediate composed of the full-length bicistronic L1 mRNA and the two proteins (ORF1p and ORF2p) encoded by that mRNA. ORF1p and ORF2p demonstrate cis-preference for their encoding mRNA. Previous studies of ORF1p, purified from bacterial and insect cells demonstrated that this protein forms trimers in vitro. While valuable for understanding ORF1p function, these in vitro approaches do not provide any information on ORF1p self-interaction in the context of mammalian cells. We used a mammalian two-hybrid (M2H) system in order to study L1 ORF1p self-interaction in human and mouse cells. We demonstrate that the M2H system successfully detects human and mouse ORF1p self-interactions in transiently transfected mammalian cells. We also generated mouse and human ORF1p-specific antibodies to characterize the expression of ORF1p fusion proteins used in the M2H system. Using these antibodies, we demonstrate that ORF1p interaction in trans leads to the formation of heterodimers that are expected to produce a positive signal in the M2H system. Although the role for L1 ORF1p cis-preference in L1 mobilization is established, the impact of ability of ORF1pto interact in trans on the L1 replication cycle is not known. Furthermore, western blot analysis of ORF1p generated by a full-length L1, wild type ORF1, or a codon-optimized ORF1 expression vector is detected in the nucleus. In contrast, the addition of a tag to the N-terminus of the mouse and human ORF1 proteins can significantly alter the subcellular localization in a tag-specific manner. These data support that nuclear localization of ORF1p may contribute to L1 (and potentially the SINE Alu) RNP nuclear access in the host cell.
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spelling pubmed-38529682013-12-09 Characterization of L1 ORF1p Self-Interaction and Cellular Localization Using a Mammalian Two-Hybrid System Sokolowski, Mark deHaro, Dawn Christian, Claiborne M. Kines, Kristine J. Belancio, Victoria P. PLoS One Research Article Long INterspersed Element-1 (LINE-1, L1) is an active retrotransposon that mobilizes using a ribonucleoprotein particle (RNP) intermediate composed of the full-length bicistronic L1 mRNA and the two proteins (ORF1p and ORF2p) encoded by that mRNA. ORF1p and ORF2p demonstrate cis-preference for their encoding mRNA. Previous studies of ORF1p, purified from bacterial and insect cells demonstrated that this protein forms trimers in vitro. While valuable for understanding ORF1p function, these in vitro approaches do not provide any information on ORF1p self-interaction in the context of mammalian cells. We used a mammalian two-hybrid (M2H) system in order to study L1 ORF1p self-interaction in human and mouse cells. We demonstrate that the M2H system successfully detects human and mouse ORF1p self-interactions in transiently transfected mammalian cells. We also generated mouse and human ORF1p-specific antibodies to characterize the expression of ORF1p fusion proteins used in the M2H system. Using these antibodies, we demonstrate that ORF1p interaction in trans leads to the formation of heterodimers that are expected to produce a positive signal in the M2H system. Although the role for L1 ORF1p cis-preference in L1 mobilization is established, the impact of ability of ORF1pto interact in trans on the L1 replication cycle is not known. Furthermore, western blot analysis of ORF1p generated by a full-length L1, wild type ORF1, or a codon-optimized ORF1 expression vector is detected in the nucleus. In contrast, the addition of a tag to the N-terminus of the mouse and human ORF1 proteins can significantly alter the subcellular localization in a tag-specific manner. These data support that nuclear localization of ORF1p may contribute to L1 (and potentially the SINE Alu) RNP nuclear access in the host cell. Public Library of Science 2013-12-04 /pmc/articles/PMC3852968/ /pubmed/24324740 http://dx.doi.org/10.1371/journal.pone.0082021 Text en © 2013 Sokolowski et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Sokolowski, Mark
deHaro, Dawn
Christian, Claiborne M.
Kines, Kristine J.
Belancio, Victoria P.
Characterization of L1 ORF1p Self-Interaction and Cellular Localization Using a Mammalian Two-Hybrid System
title Characterization of L1 ORF1p Self-Interaction and Cellular Localization Using a Mammalian Two-Hybrid System
title_full Characterization of L1 ORF1p Self-Interaction and Cellular Localization Using a Mammalian Two-Hybrid System
title_fullStr Characterization of L1 ORF1p Self-Interaction and Cellular Localization Using a Mammalian Two-Hybrid System
title_full_unstemmed Characterization of L1 ORF1p Self-Interaction and Cellular Localization Using a Mammalian Two-Hybrid System
title_short Characterization of L1 ORF1p Self-Interaction and Cellular Localization Using a Mammalian Two-Hybrid System
title_sort characterization of l1 orf1p self-interaction and cellular localization using a mammalian two-hybrid system
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3852968/
https://www.ncbi.nlm.nih.gov/pubmed/24324740
http://dx.doi.org/10.1371/journal.pone.0082021
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