Human L1 retrotransposition: insights and peculiarities learned from a cultured cell retrotransposition assay

Long Interspersed Nuclear Elements (L1s or LINEs) are the most abundant retrotransposons in the human genome, and they comprise approximately 17% of DNA. L1 retrotransposition can be mutagenic, and deleterious insertions both in the germ-line and in somatic cells have resulted in disease. Recently, an assay was developed to monitor L1 retrotransposition in cultured human cells. This assay, for the first time, now allows for a systematic study of L1 retrotransposition at the molecular level. Here, I will review progress made in L1 biology during the past three years. In general, I will limit the discussion to studies conducted on human L1s. However, interesting parallels to rodent L1s and other non-LTR retrotransposons also will be discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genomic rearrangements by LINE-1 insertion-mediated deletion in the human and chimpanzee lineages

Long INterspersed Elements (LINE-1s or L1s) are abundant non-LTR retrotransposons in mammalian genomes that are capable of insertional mutagenesis. They have been associated with target site deletions upon insertion in cell culture studies of retrotransposition. Here, we report 50 deletion events in the human and chimpanzee genomes directly linked to the insertion of L1 elements, resulting in the loss of ~18 kb of sequence from the human genome and ~15 kb from the chimpanzee genome. Our data suggest that during the primate radiation, L1 insertions may have deleted up to 7.5 Mb of target genomic sequences. While the results of our in vivo analysis differ from those of previous cell culture assays of L1 insertion-mediated deletions in terms of the size and rate of sequence deletion, evolutionary factors can reconcile the differences. We report a pattern of genomic deletion sizes similar to those created during the retrotransposition of Alu elements. Our study provides support for the existence of different mechanisms for small and large L1-mediated deletions, and we present a model for the correlation of L1 element size and the corresponding deletion size. In addition, we show that internal rearrangements can modify L1 structure during retrotransposition events associated with large deletions.     

siRNA function in RNAi: a chemical modification analysis

Various chemical modifications were created in short-interfering RNAs (siRNAs) to determine the biochemical properties required for RNA interference (RNAi). Remarkably, modifications at the 2'-position of pentose sugars in siRNAs showed the 2'-OHs were not required for RNAi, indicating that RNAi machinery does not require the 2'-OH for recognition of siRNAs and catalytic ribonuclease activity of RNA-induced silencing complexes (RISCs) does not involve the 2'-OH of guide antisense RNA. In addition, 2' modifications predicted to stabilize siRNA increased the persistence of RNAi as compared with wild-type siRNAs. RNAi was also induced with chemical modifications that stabilized interactions between A-U base pairs, demonstrating that these types of modifications may enhance mRNA targeting efficiency in allele-specific RNAi. Modifications altering the structure of the A-form major groove of antisense siRNA-mRNA duplexes abolished RNAi, suggesting that the major groove of these duplexes was required for recognition by activated RISC*. Comparative analysis of the stability and RNAi activities of chemically modified single-stranded antisense RNA and duplex siRNA suggested that some catalytic mechanism(s) other than siRNA stability were linked to RNAi efficiency. Modified or mismatched ribonucleotides incorporated at internal positions in the 5' or 3' half of the siRNA duplex, as defined by the antisense strand, indicated that the integrity of the 5' and not the 3' half of the siRNA structure was important for RNAi, highlighting the asymmetric nature of siRNA recognition for initiation of unwinding. Collectively, this study defines the mechanisms of RNAi in human cells and provides new rules for designing effective and stable siRNAs for RNAi-mediated gene-silencing applications.     

Potential for genomic instability associated with retrotranspositionally-incompetent L1 loci

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.     

The Zinc-Finger Antiviral Protein ZAP Inhibits LINE and Alu Retrotransposition

Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. To investigate the interplay between the L1 retrotransposition machinery and the host cell, we used co-immunoprecipitation in conjunction with liquid chromatography and tandem mass spectrometry to identify cellular proteins that interact with the L1 first open reading frame-encoded protein, ORF1p. We identified 39 ORF1p-interacting candidate proteins including the zinc-finger antiviral protein (ZAP or ZC3HAV1). Here we show that the interaction between ZAP and ORF1p requires RNA and that ZAP overexpression in HeLa cells inhibits the retrotransposition of engineered human L1 and Alu elements, an engineered mouse L1, and an engineered zebrafish LINE-2 element. Consistently, siRNA-mediated depletion of endogenous ZAP in HeLa cells led to a ~2-fold increase in human L1 retrotransposition. Fluorescence microscopy in cultured human cells demonstrated that ZAP co-localizes with L1 RNA, ORF1p, and stress granule associated proteins in cytoplasmic foci. Finally, molecular genetic and biochemical analyses indicate that ZAP reduces the accumulation of full-length L1 RNA and the L1-encoded proteins, yielding mechanistic insight about how ZAP may inhibit L1 retrotransposition. Together, these data suggest that ZAP inhibits the retrotransposition of LINE and Alu elements.     

Genetic Evidence That the Non-Homologous End-Joining Repair Pathway Is Involved in LINE Retrotransposition

Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs—zebrafish ZfL2-2 and human L1—in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.     

Evaluating the Extent of LINE-1 Mobility Following Exposure to Heavy Metals in HepG2 Cells

The long interspersed elements-1 (LINE1 or L1 retrotransposon) constitute 17 % of the human genome and retain mobility properties within the genome. At present, 80–100 human L1 elements are thought to be active in the genome. The mobilization of these active elements may be influenced upon exposure to the heavy metals. In the present study, we evaluated the association of aluminum, lead, and copper exposure with L1 retrotransposition in human hepatocellular carcinoma (HepG2) cell line. An in vitro retrotransposition assay using an enhanced green fluorescent protein (EGFP)-tagged L1RP cassette was established to track EGFP shining as the mark of retrotransposition. Following determination of noncytotoxic concentrations of these metals, pL1RP-EGFP-transfected HepG2 cells were subjected to long-term treatment. Flow cytometry analysis of cells treated with various concentrations of these metals along with quantitative real-time PCR was used to quantify L1 retrotransposition frequencies. Aluminum significantly increased L1 retrotransposition frequency, while no significant association was found concerning lead exposure and L1 retrotransposition. Copper treatment downregulated L1 retrotransposition as a result of EGFP-tagged L1RP expression. Our findings suggest that aluminum might have the potential to cause genomic instability by the enhancement of L1 mobilization. Thus, the risk of induced L1 retrotransposition should be considered during drug safety evaluation and risk assessments of exposure to toxic environmental agents. Further studies are needed for a more robust assay to evaluate any associations between long-term lead exposure and L1 mobility in cell culture assay.