L1 elements represent the only currently active, autonomous retrotransposon in the human genome, and they make major contributions to human genetic instability. The vast majority of the 500 000 L1 elements in the genome are defective, and only a relatively few can contribute to the retrotransposition process. However, there is currently no comprehensive approach to identify the specific loci that are actively transcribed separate from the excess of L1-related sequences that are co-transcribed within genes. We have developed RNA-Seq procedures, as well as a 1200 bp 5΄ RACE product coupled with PACBio sequencing that can identify the specific L1 loci that contribute most of the L1-related RNA reads. At least 99% of L1-related sequences found in RNA do not arise from the L1 promoter, instead representing pieces of L1 incorporated in other cellular RNAs. In any given cell type a relatively few active L1 loci contribute to the ‘authentic’ L1 transcripts that arise from the L1 promoter, with significantly different loci seen expressed in different tissues.

Expression of the L1 retrotransposon can damage the genome through insertional mutagenesis and the generation of DNA double-strand breaks (DSBs). The majority of L1 loci in the human genome are 5′-truncated and therefore incapable of retrotransposition. While thousands of full-length L1 loci remain, most are retrotranspositionally-incompetent due to inactivating mutations. However, mutations leading to premature stop codons within the L1 ORF2 sequence may yield truncated proteins that retain a functional endonuclease domain. We demonstrate that some truncated ORF2 proteins cause varying levels of toxicity and DNA damage when chronically overexpressed in mammalian cells. Furthermore, transfection of some ORF2 constructs containing premature stop codons supported low levels of Alu retrotransposition, demonstrating the potential for select retrotranspositionally-incompetent L1 loci to generate genomic instability. This result suggests yet another plausible explanation for the relative success of Alu elements in populating the human genome. Our data suggest that a subset of retrotranspositionally-incompetent L1s, previously considered to be harmless to genomic integrity, may have the potential to cause chronic DNA damage by introducing DSBs and mobilizing Alu. These results imply that the number of known L1 loci in the human genome that potentially threaten its stability may not be limited to the retrotranspositionally active loci.

Abstract-Proteins that bind to the intracellular expanses, particularly cytoplasmic tail regions, of heptahelical integral membrane receptors are of particular interest in that they can mediate or modulate trafficking or intracellular signaling.In an effort to distinguish new proteins that might promote angiotensin II type 1 (AT 1 ) receptor intracellular events, we screened a yeast 2-hybrid mouse brain library with the rat AT 1A receptor (AT 1 R) carboxyl terminus and identified GABARAP, a protein involved in intracellular trafficking of the GABA A receptor, as a binding partner for the AT 1 R. Interaction of GABARAP with the AT 1 R carboxyl terminus was further substantiated using GST pull-down assays, and binding of the full-length tagged AT 1 R to GABARAP was verified using coimmunoprecipitation. Bioluminescence resonance energy transfer assays further confirmed specific interaction of GABARAP with AT 1 R. Moreover, GABARAP clearly increased the steady-state level of plasma membrane-associated AT 1 R in PC-12 cells. Cotransfection of GABARAP with an AT 1 R fluorescent fusion protein increased PC-12 cell surface expression of the AT 1 R more than 6-fold when standardized to the level of intracellular expression. Furthermore, GABARAP overexpression in CHO-K1 cells engineered to express AT 1 R increased angiotensin II binding sites 3.7-fold and angiotensin II-induced phospho-extracellular signal-regulated kinase 1/2 and cellular proliferation significantly over levels obtained with AT 1 R overexpression alone. In addition, small interfering RNA-mediated knockdown of GABARAP reduced the steady-state levels of the AT 1 R fluorescent fusion protein by 43% and its cell surface expression by 84%. Immunoblot analyses confirmed the quantitative image data. We conclude that GABARAP binds to and promotes trafficking of the AT 1 R to the plasma membrane. receptors are 7-transmembrane G protein-coupled receptors (GPCRs) of the largest GPCR subfamily, family 1, or the rhodopsin-like family. The GPCR superfamily has more than 860 members 1 and more than 50 "GPCR-associated" proteins have now been discovered, the majority of which interact with GPCR cytoplasmic carboxyl termini. 2 Most of these are involved in trafficking, subcellular targeting, and intracellular signaling. Our preliminary studies were designed to identify proteins that bind to the cytoplasmic carboxyl terminus of the AT 1 receptor (AT 1 R), the most prevalent and best characterized of the Ang receptors. Such proteins are expected to be involved in trafficking of the AT 1 R through the secretory pathway and to the plasma membrane, as well as in ligand-mediated internalization and recycling. Moreover, our recent published studies suggest that the AT 1 R is cleaved in a ligand-dependent manner to liberate the cytoplasmic domain, a significant quantity of which traffics to the nucleus. 3 Presumably, this nuclear trafficking event also involves sequence-specific binding proteins. Using a yeast 2-hybrid (Y2H) approach to screen a mouse brain library, we have ide...

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.

Expression of long interspersed element-1 (L1) is upregulated in many human malignancies. L1 can introduce genomic instability via insertional mutagenesis and DNA double-strand breaks, both of which may promote cancer. Light exposure at night, a recently recognized carcinogen, is associated with an increased risk of cancer in shift workers. We report that melatonin receptor 1 inhibits mobilization of L1 in cultured cells through downregulation of L1 mRNA and ORF1 protein. The addition of melatonin receptor antagonists abolishes the MT1 effect on retrotransposition in a dose-dependent manner. Furthermore, melatonin-rich, but not melatonin-poor, human blood collected at different times during the circadian cycle suppresses endogenous L1 mRNA during in situ perfusion of tissue-isolated xenografts of human cancer. Supplementation of human blood with exogenous melatonin or melatonin receptor antagonist during the in situ perfusion establishes a receptor-mediated action of melatonin on L1 expression. Combined tissue culture and in vivo data support that environmental light exposure of the host regulates expression of L1 elements in tumors. Our data imply that light-induced suppression of melatonin production in shift workers may increase L1-induced genomic instability in their genomes and suggest a possible connection between L1 activity and increased incidence of cancer associated with circadian disruption.