A comprehensive analysis of recently integrated human Ta L1 elements

The Ta (transcribed, subset a) subfamily of L1 LINEs (long interspersed elements) is characterized by a 3-bp ACA sequence in the 3' untranslated region and contains approximately 520 members in the human genome. Here, we have extracted 468 Ta L1Hs (L1 human specific) elements from the draft human genomic sequence and screened individual elements using polymerase-chain-reaction (PCR) assays to determine their phylogenetic origin and levels of human genomic diversity. One hundred twenty-four of the elements amenable to complete sequence analysis were full length ( approximately 6 kb) and have apparently escaped any 5' truncation. Forty-four of these full-length elements have two intact open reading frames and may be capable of retrotransposition. Sequence analysis of the Ta L1 elements showed a low level of nucleotide divergence with an estimated age of 1.99 million years, suggesting that expansion of the L1 Ta subfamily occurred after the divergence of humans and African apes. A total of 262 Ta L1 elements were screened with PCR-based assays to determine their phylogenetic origin and the level of human genomic variation associated with each element. All of the Ta L1 elements analyzed by PCR were absent from the orthologous positions in nonhuman primate genomes, except for a single element (L1HS72) that was also present in the common (Pan troglodytes) and pygmy (P. paniscus) chimpanzee genomes. Sequence analysis revealed that this single exception is the product of a gene conversion event involving an older preexisting L1 element. One hundred fifteen (45%) of the Ta L1 elements were polymorphic with respect to insertion presence or absence and will serve as identical-by-descent markers for the study of human evolution.     

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.     

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.     

The innate antiretroviral factor APOBEC3G does not affect human LINE-1 retrotransposition in a cell culture assay

APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G) is an innate intracellular antiretroviral factor that can inhibit viral retroelements such as retroviruses and hepadnaviruses. However, it is unknown whether it can act on non-viral substrates. Retrotransposons are transposable elements that cumulatively account for about one third of the human genome. They are commonly classified in long terminal repeat (LTR) retrotransposons, which are strongly homologous to retroviruses, and non-LTR retrotransposons also known as L1 elements or LINE-1 (long interspersed nucleotide element-1) elements. Most of the L1 elements are defective and only a small number are very active in vivo, but they are responsible for nearby all of the retrotransposition in the human population. The cloning of active human L1 elements has allowed the development of tissue culture-based assays for measuring their retrotransposition potential. We used such an assay to demonstrate that APOBEC3G, which impairs the replication of exogenous retroelements, does not affect the replication of endogenous L1 retrotransposons.     

Evidence consistent with human L1 retrotransposition in maternal meiosis I

We have used a unique polymorphic 3' transduction to show that a human L1, or LINE-1 (long interspersed nucleotide element-1), retrotransposition event most likely occurred in the maternal primary oocyte during meiosis I. We characterized a truncated L1 retrotransposon with a 3' transduction that was inserted, in a Dutch male patient, into the X-linked gene CYBB, thereby causing chronic granulomatous disease. We used the unique flanking sequence to localize the precursor L1 locus, LRE3, to chromosome 2q24.1. In a cell culture assay, the retrotransposition frequency of LRE3 is greater than that for any other element that has been tested to date. The patient's mother had two LRE3 alleles that differed slightly in the 3'-flanking genomic DNA. The patient had a single LRE3 allele that was identical to one of the maternal alleles; however, the patient's insertion matched the maternal LRE3 allele that he did not inherit. Other data indicate that there is only a small chance that the father (unavailable for analysis) carries the precursor LRE3 allele. In addition, paternal origin of the insertion would have required that an LRE3 mRNA transcribed before meiosis II be carried separately from its precursor LRE3 allele in the fertilizing sperm. Since the mother carries a potential precursor allele and the insertion was on the patient's maternal X chromosome, it is highly likely that the insertion originated during maternal meiosis I.     

Human L1 retrotransposition: cis preference versus trans complementation

Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the approximately 400,000 L1s are mobile. Using a retrotransposition assay in cultured human cells, we demonstrate that L1-encoded proteins predominantly mobilize the RNA that encodes them. At much lower levels, L1-encoded proteins can act in trans to promote retrotransposition of mutant L1s and other cellular mRNAs, creating processed pseudogenes. Mutant L1 RNAs are mobilized at 0.2 to 0.9% of the retrotransposition frequency of wild-type L1s, whereas cellular RNAs are mobilized at much lower frequencies (ca. 0.01 to 0.05% of wild-type levels). Thus, we conclude that L1-encoded proteins demonstrate a profound cis preference for their encoding RNA. This mechanism could enable L1 to remain retrotransposition competent in the presence of the overwhelming number of nonfunctional L1s present in human DNA.     

LINE-1 preTa elements in the human genome

The preTa subfamily of long interspersed elements (LINEs) is characterized by a three base-pair "ACG" sequence in the 3' untranslated region, contains approximately 400 members in the human genome, and has low level of nucleotide divergence with an estimated average age of 2.34 million years old suggesting that expansion of the L1 preTa subfamily occurred just after the divergence of humans and African apes. We have identified 362 preTa L1 elements from the draft human genomic sequence, investigated the genomic characteristics of preTa L1 insertions, and screened individual elements across diverse human populations and various non-human primate species using polymerase chain reaction (PCR) assays to determine the phylogenetic origin and levels of human genomic diversity associated with the L1 elements. All of the preTa L1 elements analyzed by PCR were absent from the orthologous positions in non-human primate genomes with 33 (14%) of the L1 elements being polymorphic with respect to insertion presence or absence in the human genome. The newly identified L1 insertion polymorphisms will prove useful as identical by descent genetic markers for the study of human population genetics. We provide evidence that preTa L1 elements show an integration site preference for genomic regions with low GC content. Computational analysis of the preTa L1 elements revealed that 29% of the elements amenable to complete sequence analysis have apparently escaped 5' truncation and are essentially full-length (approximately 6kb). In all, 29 have two intact open reading frames and may be capable of retrotransposition.     

Mapping the LINE1 ORF1 protein interactome reveals associated inhibitors of human retrotransposition

LINE1s occupy 17% of the human genome and are its only active autonomous mobile DNA. L1s are also responsible for genomic insertion of processed pseudogenes and >1 million non-autonomous retrotransposons (Alus and SVAs). These elements have significant effects on gene organization and expression. Despite the importance of retrotransposons for genome evolution, much about their biology remains unknown, including cellular factors involved in the complex processes of retrotransposition and forming and transporting L1 ribonucleoprotein particles. By co-immunoprecipitation of tagged L1 constructs and mass spectrometry, we identified proteins associated with the L1 ORF1 protein and its ribonucleoprotein. These include RNA transport proteins, gene expression regulators, post-translational modifiers, helicases and splicing factors. Many cellular proteins co-localize with L1 ORF1 protein in cytoplasmic granules. We also assayed the effects of these proteins on cell culture retrotransposition and found strong inhibiting proteins, including some that control HIV and other retroviruses. These data suggest candidate cofactors that interact with the L1 to modulate its activity and increase our understanding of the means by which the cell coexists with these genomic ‘parasites’.     

LINE-like retrotransposition in Saccharomyces cerevisiae

Over one-third of human genome sequence is a product of non-LTR retrotransposition. The retrotransposon that currently drives this process in humans is the highly abundant LINE-1 (L1) element. Despite the ubiquitous nature of L1's in mammals, we still lack a complete mechanistic understanding of the L1 replication cycle and how it is regulated. To generate a genetically amenable model for non-LTR retrotransposition, we have reengineered the Zorro3 retrotransposon, an L1 homolog from Candida albicans, for use in the budding yeast Saccharomyces cerevisiae. We found that S. cerevisiae, which has no endogenous L1 homologs or remnants, can still support Zorro3 retrotransposition. Analysis of Zorro3 mutants and insertion structures suggest that this is authentic L1-like retrotransposition with remarkable resemblance to mammalian L1-mediated events. This suggests that S. cerevisiae has unexpectedly retained the basal host machinery required for L1 retrotransposition. This model will also serve as a powerful system to study the cell biology of L1 elements and for the genetic identification and characterization of cellular factors involved in L1 retrotransposition.     

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.     

MOV10 RNA Helicase Is a Potent Inhibitor of Retrotransposition in Cells

MOV10 protein, a putative RNA helicase and component of the RNA–induced silencing complex (RISC), inhibits retrovirus replication. We show that MOV10 also severely restricts human LINE1 (L1), Alu, and SVA retrotransposons. MOV10 associates with the L1 ribonucleoprotein particle, along with other RNA helicases including DDX5, DHX9, DDX17, DDX21, and DDX39A. However, unlike MOV10, these other helicases do not strongly inhibit retrotransposition, an activity dependent upon intact helicase domains. MOV10 association with retrotransposons is further supported by its colocalization with L1 ORF1 protein in stress granules, by cytoplasmic structures associated with RNA silencing, and by the ability of MOV10 to reduce endogenous and ectopic L1 expression. The majority of the human genome is repetitive DNA, most of which is the detritus of millions of years of accumulated retrotransposition. Retrotransposons remain active mutagens, and their insertion can disrupt gene function. Therefore, the host has evolved defense mechanisms to protect against retrotransposition, an arsenal we are only beginning to understand. With homologs in other vertebrates, insects, and plants, MOV10 may represent an ancient and innate form of immunity against both infective viruses and endogenous retroelements.     

A full-length and potentially active LINE element is integrated polymorphically within the <Emphasis Type="Italic">IGL</Emphasis> locus in a genomically unstable region of chromosome 22

Leukemic cells of a patient diagnosed with chronic myeloid leukemia (CML) showed a complex BCR-ABL1 rearrangement hidden within a normal appearing karyotype. Previous molecular studies had established that the 3' BCR had recombined at a novel site within the variable region of the immunoglobulin lambda locus ( IGL). A segment of DNA mapping very close to the site of the IGL/3' BCR recombination recognized a previously undescribed insertion polymorphism. A combination of molecular hybridization studies and long-range polymerase chain reaction was used to isolate a 6-kb full-length long interspersed nuclear element (LINE or L1), here designated L1(IGL), which occupies 19% of alleles in the general population. Although unclonable, DNA sequence analysis by a primer walking approach established that L1(IGL) has features characteristic of an actively retrotransposing element. The L1(IGL) element has a 5' untranslated region, two open reading frames (ORF-1 and ORF-2), a 3' untranslated region and terminates in a poly-A tail. We compared the DNA sequence and the predicted amino acid sequence of L1(IGL) with a consensus sequence compiled from seven reported active L1 elements. This analysis indicated that L1(IGL) has high potential for involvement in as yet undetermined somatically and constitutionally acquired disease, not only through recombination mechanisms, but also through retrotransposition events. This full-length L1 element maps close within the IGLlocus to L1.2, one of only nine active L1 elements that have been reported so far. L1(IGL) and L1.2 map within a wider and well-recognized region of genomic instability on chromosome 22.     

A systematic analysis of LINE-1 endonuclease-dependent retrotranspositional events causing human genetic disease

Diverse long interspersed element-1 (LINE-1 or L1)-dependent mutational mechanisms have been extensively studied with respect to L1 and Alu elements engineered for retrotransposition in cultured cells and/or in genome-wide analyses. To what extent the in vitro studies can be held to accurately reflect in vivo events in the human genome, however, remains to be clarified. We have attempted to address this question by means of a systematic analysis of recent L1-mediated retrotranspositional events that have caused human genetic disease, with a view to providing a more complete picture of how L1-mediated retrotransposition impacts upon the architecture of the human genome. A total of 48 such mutations were identified, including those described as L1-mediated retrotransposons, as well as insertions reported to contain a poly(A) tail: 26 were L1 trans-driven Alu insertions, 15 were direct L1 insertions, four were L1 trans-driven SVA insertions, and three were associated with simple poly(A) insertions. The systematic study of these lesions, when combined with previous in vitro and genome-wide analyses, has strengthened several important conclusions regarding L1-mediated retrotransposition in humans: (a) approximately 25% of L1 insertions are associated with the 3' transduction of adjacent genomic sequences, (b) approximately 25% of the new L1 inserts are full-length, (c) poly(A) tail length correlates inversely with the age of the element, and (d) the length of target site duplication in vivo is rarely longer than 20 bp. Our analysis also suggests that some 10% of L1-mediated retrotranspositional events are associated with significant genomic deletions in humans. Finally, the identification of independent retrotranspositional events that have integrated at the same genomic locations provides new insight into the L1-mediated insertional process in humans.     

The NF1 gene contains hotspots for L1 endonuclease-dependent de novo insertion

Long interspersed (L1) and Alu elements are actively amplified in the human genome through retrotransposition of their RNA intermediates by the -100 still retrotranspositionally fully competent L1 elements. Retrotransposition can cause inherited disease if such an element is inserted near or within a functional gene. Using direct cDNA sequencing as the primary assay for comprehensive NF1 mutation analysis, we uncovered in 18 unrelated index patients splicing alterations not readily explained at the genomic level by an underlying point-mutation or deletion. Improved PCR protocols avoiding allelic drop-out of the mutant alleles uncovered insertions of fourteen Alu elements, three L1 elements, and one poly(T) stretch to cause these splicing defects. Taken together, the 18 pathogenic L1 endonuclease-mediated de novo insertions represent the largest number of this type of mutations characterized in a single human gene. Our findings show that retrotransposon insertions account for as many as -0.4% of all NF1 mutations. Since altered splicing was the main effect of the inserted elements, the current finding was facilitated by the use of RNA-based mutation analysis protocols, resulting in improved detection compared to gDNA-based approaches. Six different insertions clustered in a relatively small 1.5-kb region (NF1 exons 21(16)-23(18)) within the 280-kb NF1 gene. Furthermore, three different specific integration sites, one of them located in this cluster region, were each used twice, i.e. NM_000267.3(NF1):c.1642-1_1642 in intron 14(10c), NM_000267.3(NF1):c.2835_2836 in exon 21(16), and NM_000267.3(NF1):c.4319_4320 in exon 33(25). Identification of three loci that each served twice as integration site for independent retrotransposition events as well as 1.5-kb cluster region harboring six independent insertions supports the notion of non-random insertion of retrotransposons in the human genome. Currently, little is known about which features make sites particularly vulnerable to L1 EN-mediated insertions. The here identified integration sites may serve to elucidate these features in future studies.     

Genomic deletions created upon LINE-1 retrotransposition

LINE-1 (L1) retrotransposition continues to impact the human genome, yet little is known about how L1 integrates into DNA. Here, we developed a plasmid-based rescue system and have used it to recover 37 new L1 retrotransposition events from cultured human cells. Sequencing of the insertions revealed the usual L1 structural hallmarks; however, in four instances, retrotransposition generated large target site deletions. Remarkably, three of those resulted in the formation of chimeric L1s, containing the 5' end of an endogenous L1 fused precisely to our engineered L1. Thus, our data demonstrate multiple pathways for L1 integration in cultured cells, and show that L1 is not simply an insertional mutagen, but that its retrotransposition can result in significant deletions of genomic sequence.     

Reviving the Dead: History and Reactivation of an Extinct L1

Although L1 sequences are present in the genomes of all placental mammals and marsupials examined to date, their activity was lost in the megabat family, Pteropodidae, ∼24 million years ago. To examine the characteristics of L1s prior to their extinction, we analyzed the evolutionary history of L1s in the genome of a megabat, Pteropus vampyrus, and found a pattern of periodic L1 expansion and quiescence. In contrast to the well-characterized L1s in human and mouse, megabat genomes have accommodated two or more simultaneously active L1 families throughout their evolutionary history, and major peaks of L1 deposition into the genome always involved multiple families. We compared the consensus sequences of the two major megabat L1 families at the time of their extinction to consensus L1s of a variety of mammalian species. Megabat L1s are comparable to the other mammalian L1s in terms of adenosine content and conserved amino acids in the open reading frames (ORFs). However, the intergenic region (IGR) of the reconstructed element from the more active family is dramatically longer than the IGR of well-characterized human and mouse L1s. We synthesized the reconstructed element from this L1 family and tested the ability of its components to support retrotransposition in a tissue culture assay. Both ORFs are capable of supporting retrotransposition, while the IGR is inhibitory to retrotransposition, especially when combined with either of the reconstructed ORFs. We dissected the inhibitory effect of the IGR by testing truncated and shuffled versions and found that length is a key factor, but not the only one affecting inhibition of retrotransposition. Although the IGR is inhibitory to retrotransposition, this inhibition does not account for the extinction of L1s in megabats. Overall, the evolution of the L1 sequence or the quiescence of L1 is unlikely the reason of L1 extinction.