title: Characterization of L1 ORF1p self-interaction and cellular localization using a mammalian two-hybrid system.
abstract: 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.
authors: Department of Structural and Cellular Biology, Tulane School of Medicine, Tulane Cancer Center, Tulane Center for Aging, New Orleans, Louisiana, United States of America.
authors: Mark
authors: M
authors: Sokolowski
authors: Dawn
authors: D
authors: deHaro
authors: Claiborne M
authors: CM
authors: Christian
authors: Kristine J
authors: KJ
authors: Kines
authors: Victoria P
authors: VP
authors: Belancio
mesh_terms: D016475:3T3 Cells; D000818:Animals; D006367:HeLa Cells; D006801:Humans; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics*; D008322:Mammals / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D055503:Protein Multimerization; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: C122171:Med14 protein, mouse; C531245:ORF1 protein, human; D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors