Human L1 Elements Research Articles

Long INterspersed element-1 mobility as a sensor of environmental stresses.

Long INterspersed element (LINE-1, L1) retrotransposons are the most abundant transposable elements in the human genome, constituting approximately 17%. They move by a "copy-paste" mechanism, involving reverse transcription of an RNA intermediate and insertion of its cDNA copy at a new site in the genome. L1 retrotransposition (L1-RTP) can cause insertional mutations, alter gene expression, transduce exons, and induce epigenetic dysregulation. L1-RTP is generally repressed; however, a number of observations collected over about 15 years revealed that it can occur in response to environmental stresses. Moreover, emerging evidence indicates that L1-RTP can play a role in the onset of several neurological and oncological diseases in humans. In recent years, great attention has been paid to the exposome paradigm, which proposes that health effects of an environmental factor should be evaluated considering both cumulative environmental exposures and the endogenous processes resulting from the biological response. L1-RTP could be an endogenous process considered for this application. Here, we summarize the current understanding of environmental factors that can affect the retrotransposition of human L1 elements. Evidence indicates that L1-RTP alteration is triggered by numerous and various environmental stressors, such as chemical agents (heavy metals, carcinogens, oxidants, and drugs), physical agents (ionizing and non-ionizing radiations), and experiential factors (voluntary exercise, social isolation, maternal care, and environmental light/dark cycles). These data come from in vitro studies on cell lines and in vivo studies on transgenic animals: future investigations should be focused on physiologically relevant models to gain a better understanding of this topic.

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.

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.

2013 Curt Stern Award Introduction: John Moran

2013 Curt Stern Award Introduction: John Moran

In Vitro Screening for Compounds That Enhance Human L1 Mobilization

The Long interspersed element 1 (LINE1 or L1) retrotransposon constitutes 17% of the human genome. There are currently 80–100 human L1 elements that are thought to be active in any diploid human genome. These elements can mobilize into new locations of the genome, resulting in changes in genomic information. Active L1s are thus considered to be a type of endogenous mutagen, and L1 insertions can cause disease. Certain stresses, such as gamma radiation, oxidative stress, and treatment with some agents, can induce transcription and/or mobilization of retrotransposons. In this study, we used a reporter gene assay in HepG2 cells to screen compounds for the potential to enhance the transcription of human L1. We assessed 95 compounds including genotoxic agents, substances that induce cellular stress, and commercially available drugs. Treatment with 15 compounds increased the L1 promoter activity by >1.5-fold (p<0.05) after 6 or 24 hours of treatment. In particular, genotoxic agents (benzo[a]pyrene, camptothecin, cytochalasin D, merbarone, and vinblastine), PPARα agonists (bezafibrate and fenofibrate), and non-steroidal anti-inflammatory drugs (diflunisal, flufenamic acid, salicylamide, and sulindac) induced L1 promoter activity. To examine their effects on L1 retrotransposition, we developed a high-throughput real-time retrotransposition assay using a novel secreted Gaussia luciferase reporter cassette. Three compounds (etomoxir, WY-14643, and salicylamide) produced a significant enhancement in L1 retrotransposition. This is the first study to report the effects of a wide variety of compounds on L1 transcription and retrotransposition. These results suggest that certain chemical- and drug-induced stresses might have the potential to cause genomic mutations by inducing L1 mobilization. Thus, the risk of induced L1 transcription and retrotransposition should be considered during drug safety evaluation and environmental risk assessments of chemicals.

AIM1 and LINE-1 Epigenetic Aberrations in Tumor and Serum Relate to Melanoma Progression and Disease Outcome

Aberrations in the methylation status of non-coding genomic repeat DNA sequences and specific gene promoter region are important epigenetic events in melanoma progression. Promoter methylation status in LINE-1 and Absent in melanoma-1(AIM1;6q21) associated with melanoma progression and disease outcome was assessed. LINE-1 and AIM1 methylation status was assessed in paraffin-embedded archival tissues(PEAT)(n=133) and melanoma patients’ serum(n=56). LINE-1 U-Index(hypomethylation) and AIM1 were analyzed in microdissected melanoma PEAT sections. The LINE-1 U-Index of melanoma(n=100) was significantly higher than that of normal skin(n=14) and nevi(n=12)(P=0.0004). LINE-1 U-Index level was elevated with increasing AJCC stage(P<0.0001). AIM1 promoter hypermethylation was found in higher frequency(P=0.005) in metastatic melanoma(65%) than in primary melanomas(38%). When analyzed, high LINE-1 U-Index and/or AIM1 methylation in melanomas were associated with disease-free survival(DFS) and overall survival(OS) in Stage I/II patients (P=0.017, 0.027; respectively). In multivariate analysis, melanoma AIM1 methylation status was a significant prognostic factor of OS(P=0.032). Furthermore, serum unmethylated LINE-1 was at higher levels in both stage III(n=20) and stage IV(n=36) patients compared to healthy donors(n=14)(P=0.022). Circulating methylated AIM1 was detected in patients’ serum and was predictive of OS in Stage IV patients (P=0.009). LINE-1 hypomethylation and AIM1 hypermethylation have prognostic utility in both melanoma patients’ tumors and serum.

Do human transposable element small RNAs serve primarily as genome defenders or genome regulators?

It is currently thought that small RNA (sRNA) based repression mechanisms are primarily employed to mitigate the mutagenic threat posed by the activity of transposable elements (TEs). This can be achieved by the sRNA guided processing of TE transcripts via Dicer-dependent (e.g., siRNA) or Dicer-independent (e.g., piRNA) mechanisms. For example, potentially active human L1 elements are silenced by mRNA cleavage induced by element encoded siRNAs, leading to a negative correlation between element mRNA and siRNA levels. On the other hand, there is emerging evidence that TE derived sRNAs can also be used to regulate the host genome. Here, we evaluated these two hypotheses for human TEs by comparing the levels of TE derived mRNA and TE sRNA across six tissues. The genome defense hypothesis predicts a negative correlation between TE mRNA and TE sRNA levels, whereas the genome regulatory hypothesis predicts a positive correlation. On average, TE mRNA and TE sRNA levels are positively correlated across human tissues. These correlations are higher than seen for human genes or for randomly permuted control data sets. Overall, Alu subfamilies show the highest positive correlations of element mRNA and sRNA levels across tissues, although a few of the youngest, and potentially most active, Alu subfamilies do show negative correlations. Thus, Alu derived sRNAs may be related to both genome regulation and genome defense. These results are inconsistent with a simple model whereby TE derived sRNAs reduce levels of standing TE mRNA via transcript cleavage, and suggest that human cells efficiently process TE transcripts into sRNA based on the available message levels. This may point to a widespread role for processed TE transcripts in genome regulation or to alternative roles of TE-to-sRNA processing including the mitigation of TE transcript cytotoxicity.

Reprogramming somatic cells into iPS cells activates LINE-1 retroelement mobility

Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genomic DNA and represent a major evolutionary force that has reshaped the structure and function of the human genome. However, questions remain concerning both the frequency and the developmental timing of L1 retrotransposition in vivo and whether the mobility of these retroelements commonly results in insertional and post-insertional mechanisms of genomic injury. Cells exhibiting high rates of L1 retrotransposition might be especially at risk for such injury. We assessed L1 mRNA expression and L1 retrotransposition in two biologically relevant cell types, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as well as in control parental human dermal fibroblasts (HDFs). Full-length L1 mRNA and the L1 open reading frame 1-encoded protein (ORF1p) were readily detected in hESCs and iPSCs, but not in HDFs. Sequencing analysis proved the expression of human-specific L1 element mRNAs in iPSCs. Bisulfite sequencing revealed that the increased L1 expression observed in iPSCs correlates with an overall decrease in CpG methylation in the L1 promoter region. Finally, retrotransposition of an engineered human L1 element was ~10-fold more efficient in iPSCs than in parental HDFs. These findings indicate that somatic cell reprogramming is associated with marked increases in L1 expression and perhaps increases in endogenous L1 retrotransposition, which could potentially impact the genomic integrity of the resultant iPSCs.

The Human L1 Element: A Potential Biomarker in Cancer Prognosis, Current Status and Future Directions

The discovery of new biomarkers is a rapidly advancing area in cancer biology. The challenge of biomarker development for broad clinical use requires the translation of lab-based knowledge into clinical practice. The Long Interspersed Nuclear Elements-1 (LINE-1s or L1 elements) are active members of an autonomous family of non-LTR retrotransposons and occupy nearly 17% of the human genome. There is strong experimental evidence that the global hypomethylation of genomic DNA in cancer cells results in the activation of L1s and their expression is detectable at genome, transcriptome and proteome levels in human cancer cells. Thus, human L1s constitute a potential marker for cancer cells. In this review we have attempted to scrutinize L1 expression profiles in clinical cancer studies by undertaking a comprehensive systematic analysis of papers published in the field so far with a view to providing a more complete picture of the detection methods used, improvements achieved and potential future directions. Ultimately, we will try to evaluate the potential of L1s as a molecular marker in cancer detection.

Opportunity and Means: Horizontal Gene Transfer from the Human Host to a Bacterial Pathogen

The acquisition and incorporation of genetic material between nonmating species, or horizontal gene transfer (HGT), has been frequently described for phylogenetically related organisms, but far less evidence exists for HGT between highly divergent organisms. Here we report the identification and characterization of a horizontally transferred fragment of the human long interspersed nuclear element L1 to the genome of the strictly human pathogen Neisseria gonorrhoeae. A 685-bp sequence exhibiting 98 to 100% identity to copies of the human L1 element was identified adjacent to the irg4 gene in some N. gonorrhoeae genomes. The L1 fragment was observed in ~11% of the N. gonorrhoeae population sampled but was not detected in Neisseria meningitidis or commensal Neisseria isolates. In addition, N. gonorrhoeae transcripts containing the L1 sequence were detected by reverse transcription-PCR, indicating that an L1-derived gene product may be produced. The high degree of identity between human and gonococcal L1 sequences, together with the absence of L1 sequences from related Neisseria species, indicates that this HGT event occurred relatively recently in evolutionary history. The identification of L1 sequences in N. gonorrhoeae demonstrates that HGT can occur between a mammalian host and a resident bacterium, which has important implications for the coevolution of both humans and their associated microorganisms.

Characterization of a synthetic human LINE-1 retrotransposon ORFeus-Hs

Long interspersed elements, type 1(LINE-1, L1) are the most abundant and only active autonomous retrotransposons in the human genome. Native L1 elements are inefficiently expressed because of a transcription elongation defect thought to be caused by high adenosine content in L1 sequences. Previously, we constructed a highly active synthetic mouse L1 element (ORFeus-Mm), partially by reducing the nucleotide composition bias. As a result, the transcript abundance of ORFeus-Mm was greatly increased, and its retrotransposition frequency was > 200-fold higher than its native counterpart. In this paper, we report a synthetic human L1 element (ORFeus-Hs) synthesized using a similar strategy. The adenosine content of the L1 open reading frames (ORFs) was reduced from 40% to 27% by changing 25% of the bases in the ORFs, without altering the amino acid sequence. By studying a series of native/synthetic chimeric elements, we observed increased levels of full-length L1 RNA and ORF1 protein and retrotransposition frequency, mostly proportional to increased fraction of synthetic sequence. Overall, the fully synthetic ORFeus-Hs has > 40-fold more RNA but is at most only ~threefold more active than its native counterpart (L1RP); however, its absolute retrotransposition activity is similar to ORFeus-Mm. Owing to the elevated expression of the L1 RNA/protein and its high retrotransposition ability, ORFeus-Hs and its chimeric derivatives will be useful tools for mechanistic L1 studies and mammalian genome manipulation.

Characterization of L1 retrotransposition with high-throughput dual-luciferase assays

Recent studies employing genome-wide approaches have provided an unprecedented view of the scope of L1 activities on structural variations in the human genome, and further reinforced the role of L1s as one of the major driving forces behind human genome evolution. The rapid identification of novel L1 elements by these high-throughput approaches demands improved L1 functional assays. However, the existing assays use antibiotic selection markers or fluorescent proteins as reporters; neither is amenable to miniaturization. To increase assay sensitivity and throughput, we have developed a third generation assay by using dual-luciferase reporters, in which firefly luciferase is used as the retrotransposition indicator and Renilla luciferase is encoded on the same or separate plasmid for normalization. This novel assay is highly sensitive and has a broad dynamic range. Quantitative data with high signal-to-noise ratios can be obtained from 24- up to 96-well plates in 2–4 days after transfection. Using the dual-luciferase assays, we have characterized profiles of retrotransposition by various human and mouse L1 elements, and detailed the kinetics of L1 retrotransposition in cultured cells. Its high-throughput and short assay timeframe make it well suited for routine tests as well as large-scale screening efforts.

The Impact of CpG Island on Defining Transcriptional Activation of the Mouse L1 Retrotransposable Elements

BackgroundL1 retrotransposable elements are potent insertional mutagens responsible for the generation of genomic variation and diversification of mammalian genomes, but reliable estimates of the numbers of actively transposing L1 elements are mostly nonexistent. While the human and mouse genomes contain comparable numbers of L1 elements, several phylogenetic and L1Xplore analyses in the mouse genome suggest that 1,500–3,000 active L1 elements currently exist and that they are still expanding in the genome. Conversely, the human genome contains only 150 active L1 elements. In addition, there is a discrepancy among the nature and number of mouse L1 elements in L1Xplore and the mouse genome browser at the UCSC and in the literature. To date, the reason why a high copy number of active L1 elements exist in the mouse genome but not in the human genome is unknown, as are the potential mechanisms that are responsible for transcriptional activation of mouse L1 elements.Methodology/Principal FindingsWe analyzed the promoter sequences of the 1,501 potentially active mouse L1 elements retrieved from the GenBank and L1Xplore databases and evaluated their transcription factors binding sites and CpG content. To this end, we found that a substantial number of mouse L1 elements contain altered transcription factor YY1 binding sites on their promoter sequences that are required for transcriptional initiation, suggesting that only a half of L1 elements are capable of being transcriptionally active. Furthermore, we present experimental evidence that previously unreported CpG islands exist in the promoters of the most active TF family of mouse L1 elements. The presence of sequence variations and polymorphisms in CpG islands of L1 promoters that arise from transition mutations indicates that CpG methylation could play a significant role in determining the activity of L1 elements in the mouse genome.ConclusionsA comprehensive analysis of mouse L1 promoters suggests that the number of transcriptionally active elements is significantly lower than the total number of full-length copies from the three active mouse L1 families. Like human L1 elements, the CpG islands and potentially the transcription factor YY1 binding sites are likely to be required for transcriptional initiation of mouse L1 elements.

L1 retrotransposition occurs mainly in embryogenesis and creates somatic mosaicism

Long Interspersed Element 1 (L1) is a retrotransposon that comprises approximately 17% of the human genome. Despite its abundance in mammalian genomes, relatively little is understood about L1 retrotransposition in vivo. To study the timing and tissue specificity of retrotransposition, we created transgenic mouse and rat models containing human or mouse L1 elements controlled by their endogenous promoters. Here, we demonstrate abundant L1 RNA in both germ cells and embryos. However, the integration events usually occur in embryogenesis rather than in germ cells and are not heritable. We further demonstrate L1 RNA in preimplantation embryos lacking the L1 transgene and L1 somatic retrotransposition events in blastocysts and adults lacking the transgene. Together, these data indicate that L1 RNA transcribed in male or female germ cells can be carried over through fertilization and integrate during embryogenesis, an interesting example of heritability of RNA independent of its encoding DNA. Thus, L1 creates somatic mosaicism during mammalian development, suggesting a role for L1 in carcinogenesis and other disease.

Epigenetic control of mammalian LINE-1 retrotransposon by retinoblastoma proteins

Long interspersed nuclear elements (LINEs or L1 elements) are targeted for epigenetic silencing during early embryonic development and remain inactive in most cells and tissues. Here we show that E2F–Rb family complexes participate in L1 elements epigenetic regulation via nucleosomal histone modifications and recruitment of histone deacetylases (HDACs) HDAC1 and HDAC2. Our experiments demonstrated that (i) Rb and E2F interact with human and mouse L1 elements, (ii) L1 elements are deficient in both heterochromatin-associated histone marks H3 tri methyl K9 and H4 tri methyl K20 in Rb family triple knock out (Rb, p107, and p130) fibroblasts (TKO), (iii) L1 promoter exhibits increased histone H3 acetylation in the absence of HDAC1 and HDAC2 recruitment, (iv) L1 expression in TKO fibroblasts is upregulated compared to wild type counterparts, (v) L1 expression increases in the presence of the HDAC inhibitor TSA. On the basis of these findings we propose a model in which L1 sequences throughout the genome serve as centers for heterochromatin formation in an Rb family-dependent manner. As such, Rb proteins and L1 elements may play key roles in heterochromatin formation beyond pericentromeric chromosomal regions. These findings describe a novel mechanism of L1 reactivation in mammalian cells mediated by failure of corepressor protein recruitment by Rb, loss of histone epigenetic marks, heterochromatin formation, and increased histone H3 acetylation.

The impact of multiple splice sites in human L1 elements

LINE-1 elements represent a significant proportion of mammalian genomes. The impact of their activity on the structure and function of the host genomes has been recognized from the time of their discovery as an endogenous source of insertional mutagenesis. L1 elements contain numerous functional internal polyadenylation signals and splice sites that generate a variety of processed L1 transcripts. These sites are also reported to contribute to the generation of hybrid transcripts between L1 elements and host genes. Using northern blot analysis we demonstrate that L1 splicing, but not L1 polyadenylation, is delayed during the course of L1 expression. L1 splicing can also be negatively regulated by EBV SM protein known to alter this process. These results suggest a potential for L1 mRNA processing to be regulated in a tissue- and/or development-specific manner. The delay in L1 splicing may also serve to protect host genes from the excessive burden of L1 interference with their normal expression via aberrant splicing.

A yeast model for target-primed (non-LTR) retrotransposition

BackgroundTarget-primed (non-LTR) retrotransposons, such as the human L1 element, are mobile genetic elements found in many eukaryotic genomes. They are often present in large numbers and their retrotransposition can cause mutations and genomic rearrangements. Despite their importance, many aspects of their replication are not well understood.ResultsWe have developed a yeast model system for studying target-primed retrotransposons. This system uses the Zorro3 element from Candida albicans. A cloned copy of Zorro3, tagged with a retrotransposition indicator gene, retrotransposes at a high frequency when introduced into an appropriate C. albicans host strain. Retrotransposed copies of the tagged element exhibit similar features to the native copies, indicating that the natural retrotransposition pathway is being used. Retrotransposition is dependent on the products of the tagged element's own genes and is highly temperature-regulated. The new assay permits the analysis of the effects of specific mutations introduced into the cloned element.ConclusionThis Zorro3 retrotransposition assay system complements previously available target-primed retrotransposition assays. Due to the relative simplicity of the growth, manipulation and analysis of yeast cells, the system should advance our understanding of target-primed retrotransposition.

APOBEC3 Proteins Inhibit Human LINE-1 Retrotransposition

The human cytidine deaminase family APOBEC3 represents a novel group of proteins in the field of innate defense mechanisms that has been shown to be active against a variety of retroviruses. Here we examined whether members of the APO-BEC3 family have an impact on retrotransposition of human long interspersed nuclear elements (LINE-1s or L1s). Using a retrotransposition reporter assay in HeLa cells, we demonstrate that in the presence of transiently transfected APOBEC3A, L1 retrotransposition frequency was reduced by up to 85%. Although APOBEC3G and -3H did not influence L1 retrotransposition notably, expression of APOBEC3B, -3C, and -3F inhibited transposition by approximately 75%. Although reverse transcription of L1s occurs in the nucleus and APOBEC3 proteins are believed to act via DNA deamination during reverse transcription, activity against L1 retrotransposition was not correlated with nuclear localization of APOBEC3s. We demonstrate that APOBEC3C and APOBEC3B were endogenously expressed in HeLa cells. Accordingly, down-regulation of APOBEC3C by RNA interference enhanced L1 retrotransposition by approximately 78%. Sequence analyses of de novo L1 retrotransposition events that occurred in the presence of overexpressed APOBEC3 proteins as well as the analyses of pre-existing endogenous L1 elements did not reveal an enhanced rate of G-to-A transitions, pointing to a mechanism independent of DNA deamination. This study presents evidence for a role of host-encoded APOBEC3 proteins in the regulation of L1 retrotransposition.

L1 retrotransposition in nondividing and primary human somatic cells

Whether long interspersed element-1 (L1 or LINE-1) retrotransposition can occur in quiescent, nondividing, and/or terminally differentiated somatic cells has remained an unanswered fundamental question in human genetics. Here, we used a ubiquitously active phosphoglycerate kinase-1 promoter to drive the expression of a highly active human L1 element from an adenovirus-L1 hybrid vector. This vector system achieved retrotransposition in up to 91% of actively growing immortalized cells, and we demonstrated that L1 retrotransposition can be suppressed by the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine. This adenovirus vector enabled efficient delivery of the L1 element into differentiated primary human somatic cells and G1/S-arrested cells, resulting in retrotransposition in both cases; however, it was not detected in G0-arrested cells. Thus, these data indicate that L1 retrotransposition can occur in nondividing somatic cells.

LINE-1 RNA splicing and influences on mammalian gene expression

Long interspersed element-1 elements compose on average one-fifth of mammalian genomes. The expression and retrotransposition of L1 is restricted by a number of cellular mechanisms in order to limit their damage in both germ-line and somatic cells. L1 transcription is largely suppressed in most tissues, but L1 mRNA and/or proteins are still detectable in testes, a number of specific somatic cell types, and malignancies. Down-regulation of L1 expression via premature polyadenylation has been found to be a secondary mechanism of limiting L1 expression. We demonstrate that mammalian L1 elements contain numerous functional splice donor and acceptor sites. Efficient usage of some of these sites results in extensive and complex splicing of L1. Several splice variants of both the human and mouse L1 elements undergo retrotransposition. Some of the spliced L1 mRNAs can potentially contribute to expression ofopen reading frame 2-related products and therefore have implications for the mobility of SINEs even if they are incompetent for L1 retrotransposition. Analysis of the human EST database revealed that L1 elements also participate in splicing events with other genes. Such contribution of functional splice sites by L1 may result in disruption of normal gene expression or formation of alternative mRNA transcripts.