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

Feedback inhibition of L1 and alu retrotransposition through altered double strand break repair kinetics

<p>Abstract</p> <p>Background</p> <p>Cells adapt to various chronic toxic exposures in a multitude of ways to minimize further damage and maximize their growth potential. Expression of L1 elements in the human genome can be greatly deleterious to cells, generating numerous double strand breaks (DSBs). Cells have been reported to respond to chronic DSBs by altering the repair of these breaks, including increasing the rate of homology independent DSB repair. Retrotransposition is strongly affected by proteins involved in DSB repair. Therefore, L1 expression has the potential to be a source of chronic DSBs and thus bring about the changes in cellular environment that could ultimately restrict its own retrotransposition.</p> <p>Results</p> <p>We demonstrate that constitutive L1 expression leads to quicker DSB repair and decreases in the retrotransposition potential of L1 and other retrotransposons dependent on L1 expression for their mobility. This cellular adaptation results in reduced sensitivity to L1 induced toxicity. These effects can be induced by constitutive expression of the functional L1 ORF2 alone, but not by the constitutive expression of an L1 open reading frame 2 with mutations to its endonuclease and reverse transcriptase domains. This adaptation correlates with the relative activity of the L1 introduced into the cells.</p> <p>Conclusions</p> <p>The increased number of DSBs resulting from constitutive expression of L1 results in a more rapid rate of repair. The cellular response to this L1 expression also results in attenuation of retrotransposition and reduced sensitivity of the cells to negative consequences of L1 ORF2 expression. The influence does not appear to be through RNA interference. We believe that the increased rate of DSB repair is the most likely cause of the attenuation of retrotransposition. These alterations act as a fail safe mechanism that allows cells to escape the toxicity associated with the unchecked L1 expression. This gives cells that overexpress L1, such as tumor cells, the ability to survive the high levels of expression. However, the increased rate of break repair may come at the cost of accuracy of repair of the lesion, resulting in increased genomic instability.</p

Somatic expression of LINE-1 elements in human tissues

LINE-1 expression damages host DNA via insertions and endonuclease-dependent DNA double-strand breaks (DSBs) that are highly toxic and mutagenic. The predominant tissue of LINE-1 expression has been considered to be the germ line. We show that both full-length and processed L1 transcripts are widespread in human somatic tissues and transformed cells, with significant variation in both L1 expression and L1 mRNA processing. This is the first demonstration that RNA processing is a major regulator of L1 activity. Many tissues also produce translatable spliced transcript (SpORF2). An Alu retrotransposition assay, COMET assays and 53BP1 foci staining show that the SpORF2 product can support functional ORF2 protein expression and can induce DNA damage in normal cells. Tests of the senescence-associated β-galactosidase expression suggest that expression of exogenous full-length L1, or the SpORF2 mRNA alone in human fibroblasts and adult stem cells triggers a senescence-like phenotype, which is one of the reported responses to DNA damage. In contrast to previous assumptions that L1 expression is germ line specific, the increased spectrum of tissues exposed to L1-associated damage suggests a role for L1 as an endogenous mutagen in somatic tissues. These findings have potential consequences for the whole organism in the form of cancer and mammalian aging

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

Regulation of L1 expression and retrotransposition by melatonin and its receptor: implications for cancer risk associated with light exposure at night.

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

Many LINE1 elements contribute to the transcriptome of human somatic cells

Over 600 LINE 1 elements are shown to be transcribed in humans; 400 of these are full-length elements in the reference genome

The RNA Polymerase Dictates ORF1 Requirement and Timing of LINE and SINE Retrotransposition

Mobile elements comprise close to one half of the mass of the human genome. Only LINE-1 (L1), an autonomous non-Long Terminal Repeat (LTR) retrotransposon, and its non-autonomous partners—such as the retropseudogenes, SVA, and the SINE, Alu—are currently active human retroelements. Experimental evidence shows that Alu retrotransposition depends on L1 ORF2 protein, which has led to the presumption that LINEs and SINEs share the same basic insertional mechanism. Our data demonstrate clear differences in the time required to generate insertions between marked Alu and L1 elements. In our tissue culture system, the process of L1 insertion requires close to 48 hours. In contrast to the RNA pol II-driven L1, we find that pol III transcribed elements (Alu, the rodent SINE B2, and the 7SL, U6 and hY sequences) can generate inserts within 24 hours or less. Our analyses demonstrate that the observed retrotransposition timing does not dictate insertion rate and is independent of the type of reporter cassette utilized. The additional time requirement by L1 cannot be directly attributed to differences in transcription, transcript length, splicing processes, ORF2 protein production, or the ability of functional ORF2p to reach the nucleus. However, the insertion rate of a marked Alu transcript drastically drops when driven by an RNA pol II promoter (CMV) and the retrotransposition timing parallels that of L1. Furthermore, the “pol II Alu transcript” behaves like the processed pseudogenes in our retrotransposition assay, requiring supplementation with L1 ORF1p in addition to ORF2p. We postulate that the observed differences in retrotransposition kinetics of these elements are dictated by the type of RNA polymerase generating the transcript. We present a model that highlights the critical differences of LINE and SINE transcripts that likely define their retrotransposition timing