Although human diseases of retrotransposition-derived etiology have been documented, retrotransposon RNA expression and the occurrence of retrotransposition events in the human oocyte are not studied. We investigated the RNA expression of L1 and HERV-K10 retrotransposons in human oocytes by RT–PCR analysis with designed primers. Using denucleated germinal vesicles (GVs), we detected RT–PCR products of expressed L1, HERV-K10 and, unexpectedly, SINE-R, VNTR and Alu (SVA) retrotransposons. Their transcript specificities were identified as such following RNA-FISH and their origin by cloning and sequence alignment analyses. Assessing the expression level in comparison with somatic cells by densitometry analysis, we found that although in normal lymphocytes and transformed HeLa cells their profile was in an order of L1 > HERV-K10 > SVA, remarkably this was reversed in oocytes. To investigate whether de novo retrotransposition events occur and reverse transcriptases are expressed in the human oocyte, we introduced in GVs either a retrotransposition active human L1 or mouse reverse transcriptase deficient-VL30 retrotransposon tagged with an EGFP-based retrotransposition cassette. Interestingly, in both the cases, we observed EGFP-positive oocytes, associated with an abnormal morphology for L1 and granulation for VL30, and the retrotransposition events were confirmed by PCR. Our results: (i) show that L1, HERV-K10 and SVA retrotransposons are transcriptionally expressed and (ii) provide evidence, for the first time, for retrotransposition events occurring in the human oocyte. These findings suggest that both, network of retrotransposon transcripts and controlled retrotranspositions, might serve important functions required for oocyte development and fertilization while the uncontrolled ones might explain the onset of genetic disorders.

The impact of long terminal repeat (LTR) retrotransposition on cell fate is unknown. Here, we investigated the effect of VL30 retrotransposition on cell death in SV40-transformed mouse SVTT1 cells. Transfection of a VL30 retrotransposon decreased the clonogenicity of SVTT1 by 17-fold, as compared to parental NIH3T3 cells. Correlated levels of retrotransposition frequency and cell death rates were found in retrotransposition-positive SVTT1 cloned cells, exhibiting DNA fragmentation, nuclear condensation, multinucleation and cytoplasmic vacuolization. Analysis of activation of effector caspases revealed a caspase-independent cell death mechanism. However, cell death was associated with p53 induction and concomitant upregulation of PUMAα and Bax and downregulation of Bcl-2 and Hsp70 protein expression. Moreover, we found partial loss of colocalization of large T-antigen (LT)/p53 and p53 translocation to mitochondria, leading to mitochondrial outer membrane permeabilization (MOMP) accompanied by lysosomal membrane permeabilization (LMP). Interestingly, treatment with the antioxidant N-acetylcysteine abolished cell death, suggesting the involvement of mitochondrial-derived reactive oxygen species, and resulted in an increase of retrotransposition frequency. Importantly, the induction of cell death was VL30 retrotransposon-specific as VL30 mobilization was induced; in contrast, mobilization of the non-LTR L1 (LINE-1, long interspersed nuclear element-1), B2 and LTR MusD retrotransposons decreased. Our results provide, for the first time, strong evidence that VL30 retrotransposition mediates cell death via mitochondrial and lysosomal damage, uncovering the role of retrotransposition as a nuclear signal activating a mitochondrial-lysosomal crosstalk in triggering cell death.

Retrotransposons are mobile elements that have a high impact on shaping the mammalian genomes. Since the availability of whole genomes, genomic analyses have provided novel insights into retrotransposon biology. However, many retrotransposon families and their possible genomic impact have not yet been analysed. Here, we analysed the structural features, the genomic distribution and the evolutionary history of mouse VL30 LTR-retrotransposons. In total, we identified 372 VL30 sequences categorized as 86 full-length and 49 truncated copies as well as 237 solo LTRs, with non-random chromosomal distribution. Full-length VL30s were highly conserved elements with intact retroviral replication signals, but with no protein-coding capacity. Analysis of LTRs revealed a high number of common transcription factor binding sites, possibly explaining the known inducible and tissue-specific expression of individual elements. The overwhelming majority of full-length and truncated elements (82/86 and 40/49, respectively) contained one or two specific motifs required for binding of the VL30 RNA to the poly-pyrimidine tract-binding protein-associated splicing factor (PSF). Phylogenetic analysis revealed three VL30 groups with the oldest emerging ~17.5 Myrs ago, while the other two were characterized mostly by new genomic integrations. Most VL30 sequences were found integrated either near, adjacent or inside transcription start sites, or into introns or at the 3′ end of genes. In addition, a significant number of VL30s were found near Krueppel-associated box (KRAB) genes functioning as potent transcriptional repressors. Collectively, our study provides data on VL30s related to their: (a) number and structural features involved in their transcription that play a role in steroidogenesis and oncogenesis; (b) evolutionary history and potential for retrotransposition; and (c) unique genomic distribution and impact on gene expression.

Arsenic is an environmental contaminant with known cytotoxic and carcinogenic properties, but the cellular mechanisms of its action are not fully known. As retrotransposition consists a potent mutagenic factor affecting genome stability, we investigated the effect of arsenic on retrotransposition of an enhanced green fluorescent protein (EGFP)–tagged nonautonomous long terminal repeat (LTR)-retrotransposon viral-like 30 (VL30) in a mouse NIH3T3 cell culture-retrotransposition assay. Flow cytometry analysis of assay cells treated with 2.5–20μM sodium arsenite revealed induction of retrotransposition events in a dose- and time-dependent manner, which was further confirmed as genomic integrations by PCR analysis and appearance of EGFP-positive cells by UV microscopy. Specifically, 20μM sodium arsenite strongly induced the VL30 retrotransposition frequency, which was ~90,000-fold higher than the natural one and also VL30 RNA expression was ~6.6-fold. Inhibition of the activity of endogenous reverse transcriptases by efavirenz at 15μM or nevirapine at 375μM suppressed the arsenite-induced VL30 retrotransposition by 71.16 or 79.88%, respectively. In addition, the antioxidant N-acetyl-cysteine reduced the level of arsenite-induced retrotransposition, which correlated with the rescue of arsenite-induced G2/M cell cycle arrest and cell toxicity. Treatment of assay cells ectopically overexpressing the human heat-shock protein 70 (Hsp70) with 15μM sodium arsenite resulted in an additional ~4.5-fold induction of retrotransposition compared with normal assay cells, whereas treatment with 20μM produced a massive cell death. Our results show for the first time that arsenic both as an oxidative and heat-shock mimicking agent is a potent inducer of VL30 retrotransposition in mouse cells. The impact of arsenic-induced retrotransposition, as a cellular response, on contribution to or explanation of the arsenic-associated toxicity and carcinogenicity is discussed.

Retroviral reverse transcriptase (RT) plays a definite role in retroviral life cycle and is essential for the process of retrotransposition. We investigated the RNA expression of endogenous reverse transcriptases (enRTs) in the NIH3T3 mouse genome using, as a probe, a mixture of RT-PCR generated reverse transcriptase products potentially detecting a large number of RTs following treatment with different agents. We found that the expression of enRTs is induced approximately 500-fold following 5'-azacytidine-treatment. Amongst steroid hormones used such as estradiol, diethylstilbestrol, progesterone and dexamethasone only the latter was effective in inducing enRTs up to 4-fold at a concentration of 10(-7) M. Expression of a mouse dominant-negative form of p53 protein in cell clones resulted in induction of 20- to 50-fold, whereas C2-ceramide in a 4-fold induction at concentrations of 20-80 micro M. In a parallel analysis, the respective expression of the transposable viral-like 30 elements (VL30s) was also measured. Their expression was induced up to 50-fold by 5'-azacytidine, overexpression of the p53 gene and C2-ceramide at 80 micro M. It was also induced approximately 3- to 5-fold following estradiol, diethylstilbestrol or progesterone treatment and 30-fold by dexamethasone. Collectively, our results suggest that such stimuli inducing enRTs might play a role in the activation of transcription and retrotransposition of VL30.