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.

Development of a monoclonal antibody specific to the endonuclease domain of the human LINE-1 ORF2 protein

“…Similar to previously reported polyclonal antibodies [27], our monoclonal anti-ORF2p antibody detects untagged ORF2 protein expressed from the plasmids containing fulllength wild-type or codon-optimized L1 elements. This characteristic is beneficial because the addition of different tags can interfere with L1 protein function or subcellular localization [20,51]. Using bacterially purified endonuclease protein, we generated a standard curve that allowed us to determine the sensitivity of our monoclonal antibodies, which is about 10 ng of the purified protein under the described detection conditions ( Figure 6).…”

Involvement of Conserved Amino Acids in the C-Terminal Region of LINE-1 ORF2p in Retrotransposition

“…After 3 hr the transfection reaction was replaced with serum containing HeLa media. The cells were harvested 24 hr later using total protein extraction protocol (Sokolowski et al 2013).…”

LINE-1 and the cell cycle: protein localization and functional dynamics

“…In HeLa cells, this observation was made more evident by the fact that daughter cells displaying nuclear ORF1p were sometimes connected by intercellular bridges often containing filaments of DNA and persisting from incomplete cytokinesis during the previous mitosis ( Fig. 1D, lower panels) (Steigemann et al, 2009, Carlton et al, 2012. To quantify our initial qualitative observation of closer cell proximity for cells displaying nuclear ORF1p, we employed automated image acquisition of HeLa cells expressing recoded L1 and stained for ORF1p (see "quantification and statistical analysis" section).…”

“…L1 RNPs allegedly accumulate in cytoplasmic stress granules (SGs) and processing bodies (PBs) (Goodier et al, 2007, Hu et al, 2015, but the role of these cellular structures in the L1 lifecycle is still controversial. L1 RNPs accumulate mainly in the cytoplasm but nuclear ORF1p was also observed in a certain percentage of cells using cell lines and cancer specimens (Sokolowski et al, 2013, Sharma et al, 2016, Doucet et al, 2016, Goodier et al, 2007, Harris et al, 2010. While several specific antibodies against ORF1p have been raised (Taylor et al, 2013, Wylie et al, 2016, Doucet-O'Hare et al, 2015, a highly effective antibody against ORF2 protein that would allow definitive observation of protein localization is still lacking.…”

“…LINE-1 encoded proteins enter the nucleus during mitosis and retrotransposition appears to occur mainly during S phase. (Doucet et al, 2016, Luo et al, 2016, Sokolowski et al, 2013, Goodier et al, 2007, Goodier et al, 2004, Sharma et al, 2016, Rodic et al, 2014, Taylor et al, 2013, Branciforte and Martin, 1994 have shown varying localization of ORF1 protein in cells growing in culture or in mammalian specimens from various organs and tumors. In particular, the nuclear localization of LINE-1 encoded proteins has been sparsely studied and the mechanisms driving import of L1 retrotransposition intermediates into the nucleus are largely unknown.…”