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. 2010 Jul;38(12):3909-22.
doi: 10.1093/nar/gkq132. Epub 2010 Mar 9.

Somatic expression of LINE-1 elements in human tissues

Victoria P Belancio  1 , Astrid M Roy-Engel, Radhika R Pochampally, Prescott Deininger
Affiliations

Somatic expression of LINE-1 elements in human tissues

Victoria P Belancio et al. Nucleic Acids Res. 2010 Jul.

Abstract

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 beta-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.

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Figures

Figure 1.
Figure 1.
Endogenous L1 expression in normal human tissues. (A) A schematic representation of the L1 structure and some transcription products detected in normal tissues and cancer cells. Full-length L1 (FL1) element contains a 5′-UTR, two open reading frames 1 and 2 (ORF1 and 2), 3′-UTR and a polyadenylation signal (pA). L1 transcription results in the production of the full-length L1 (FL1) mRNA, prematurely polyadenylated mRNAs (pA), and spliced and polyadenylated mRNAs (Sp), one of which has the potential to express L1 ORF2 protein alone (SpORF2) as confirmed by its capability to mobilize Alu in a tissue culture assay (see Figure 4B). Dashed lines correspond to the L1 sequences that are removed by splicing. Horizontal arrows indicate relative positions of the strand-specific 5′-UTR 100 bp and 5′-UTR 600 bp probes used for northern blot analysis in this study. (B–E) Northern blot analysis of the endogenous L1 expression in various adult human tissues. Es, esophagus; St, stomach; Col, colon; SM, skeletal muscle; HM, heart muscle; Cer, cervix; Adr, adrenal gland; Kid, kidney; Spl, spleen; Pan, pancreas; Br, brain; Ts, testis; Ov, ovaries; Pl, placenta; LN, lung; PR, prostate; TH, thymus; LV, liver. The very left lane shows L1.3 wt (L1.3) and mutant (L1.3 M) that contains a mutation in the strongest polyA site (15) expression profiles in NIH 3T3 cells transiently transfected with these human L1 expression vectors. L1 transcription results in the production of the full-length L1 (FL1) mRNA, prematurely polyadenylated mRNAs (pA), and spliced and polyadenylated mRNAs (Sp) one of which has the potential to express L1 ORF2 protein (SpORF2). Actin denotes β-actin mRNA detected with a strand-specific probe.
Figure 2.
Figure 2.
Endogenous L1 expression in human adult stem cells and cancer cell lines. (A) Schematic of the full-length L1 element (FL1) and the ORF2 splice product (SpORF2). Arrows indicate the positions of the RNA strand-specific probes. (B) The left panel represents northern blot analysis of the RNA profiles of endogenously expressed L1 elements in human cervical (HeLa) and breast (MCF7) cell lines and human mesenchymal stem cells (MSCs) using the 5′-UTR 100 bp RNA strand-specific probe. The right panel is the same northern blots probed with the 5′-UTR 600 bp strand-specific RNA probe that detects polyadenylated, but not spliced, L1 mRNA. See Supplementary Figure S1B for the northern blot with the ORF2 probe. FL1, pA, Sp, SpORF2 and actin are marked as described in Figure 1. Asterisks mark bands specific to human MSCs. (C) Relative L1 expression and processing in MCF7 and HeLa cells. Black bars indicate the amount of the full-length L1 mRNA as a percentage of the total L1-related products detected in each cell line with the 5′-UTR 100 bp probe (scale shown at the left y-axis). Gray bars represent expression of all L1-related transcripts detected in each cell type relative to actin mRNA expression (scale shown at the right y-axis). Note that even though both cell lines express similar steady-state levels of the total L1 mRNAs, the full-length L1 mRNA composes only 10% of the total L1 products in HeLa cells compared to over 50% in MCF7 cells.
Figure 3.
Figure 3.
Various adult human tissues support production of the spliced L1 transcript that has a potential to produce ORF2 protein. (A) Schematic of the full-length L1 element (FL1) and the spliced ORF2 transcript (SpORF2). Arrows indicate the positions of the RT–PCR primers. (B) RT–PCR analysis of the L1 splice products in human Ts, testis; Ov, ovary; Pl, placenta; Ad, adrenal gland. RT(+) and RT(−) panels indicate reactions with and without RT, respectively. 100 M, 100 bp marker. The two differentially migrating bands represent L1 splice products utilizing various L1 splice donor and acceptor sites (indicated on the left).
Figure 4.
Figure 4.
SpORF2 splice product can produce functional L1 ORF2 protein. (A) Experimental approach for L1 ORF2-dependent Alu retroposition. An Alu element (gray box) tagged with a backwards neomycin resistance (NeoR) gene (22) (white arrow indicating the orientation of NeoR transcription) expression of which is driven by a promoter (black box marked with P) in the orientation opposite of the Alu transcription. NeoR gene ORF is interrupted by a self-splicing intron that is removed upon Alu transcription and a functional NeoR gene can be expressed upon tagged Alu retroposition. The tagged Alu expression cassette is transfected into HeLa cells with the wt L1, SpORF2 expression vectors or tagged Alu vector alone, which detects ‘background activity’ generated by the endogenously expressed L1 ORF2. (B) SpORF2 L1 RNA products can produce functional ORF2 protein to drive Alu retroposition in tissue culture. A mean± SD of NeoR colonies for each construct is shown (see Supplementary Figure S3A for construct design and Supplemenatry Figure S3B for additional controls). (C) Transient expression of SpORF2 splice product in HeLa cells leads to DNA damage. HeLa cells were transiently transfected with the empty or SpORF2 expression vector, or subjected to 5 Gy of IR. Neutral COMET assay was performed and a mean± SD of the tail extent moment from three independent experiments is shown. Asterisks indicate statistically significant difference between the treatments and the control empty vector (t-test, P < 0.05).
Figure 5.
Figure 5.
Endogenously expressed L1 ORF2 supports exogenous Alu retroposition in HeLa cells. (A) Effect of d4T on the neomycin resistant colony formation. HeLa cells transiently transfected with a neomycin tagged Alu supplemented with non-tagged L1 (Alu+L1), a tagged L1 (L1) or an unrelated plasmid containing neomycin resistance gene (Neo control) were treated with different doses of 2′,3′-didehydro-3′-deoxy-thymidine, d4T, (0–50 µM) for 7 days. Following the treatment, cells were grown in the presence of G418 for 14 days, after which the NeoR colonies were stained and counted. The results from the untreated (0 µM) cells were arbitrarily assigned as 100% (dashed line). The relative percentage means ± SD are shown. The total number of colonies observed for the different dose treatments are indicated above each column. No significant effect on the colony formation was detected for the d4T treated control (neo control) when compared to the untreated sample (P > 0.5, paired t-test). Both tested d4T doses significantly affected the ability of the tagged L1 and Alu vectors to generate Neo resistant colonies through the retrotransposition process (P ≤ 0.03, paired t-test astersik). (B) Schematic representation of the Alu expression vector (gray rectangle) tagged with neomycin gene (white arrow) interrupted by a self-spliced intron (SSI). P designates promoter that drives functional Neo gene expression only after successful retroposition. Black arrows indicate relative positions of the primers used in PCR amplification specifically designed to distinguish between a spliced retrotransposed insert and the original unspliced vector sequence [(AluNeo(+) and AluNeoEx1(−)]. Primer sequences are shown in Supplementary Figure S5. To increase specificity, the upstream primer is specific to the Alu construct, so that this pair of primers will not amplify NeoR gene present in other expression vectors to eliminate detection of any potential contamination from the neo/kan-expressing plasmids (Supplementary Figure S4). (C) PCR analysis of DNA recovered from the neomycin resistance (NeoR) colonies generated in HeLa cells without supplementation with the exogenous L1 expression vector. The size of the generated PCR product corresponds to the spliced NeoR gene in the Alu expression vector. AV is Alu expression plasmid (positive control for unspliced product). NV is non-Alu neomycin expression vector (negative control for specific Alu amplification). NC is negative control (PCR reaction without DNA template). M is DNA marker.
Figure 6.
Figure 6.
L1 and SpORF2 expression in normal human cells results in DNA damage. (A) Normal human fibroblasts transiently transfected with the empty vector or vectors expressing L1 or SpORF2 were analyzed for 53BP1 foci formation 21 h post-transfection. 53BP1 is one of the characterized responders recruited to the sites of DNA DSBs and it is commonly used as a marker for DNA DSBs. Treatment with 1.85 mM of H2O2 for 10 min was used as positive control. Overlay of Hoechst and antip53BP antibodies is shown. Arrows indicate cells with distinct staining foci. (B) Quantitation of the 53BP1 foci positive cells as a percent of the total cells (mean ± SD). Asterisks indicate significant difference determined by paired t-test with P = 0.024, 0.014, 0.001 for L1, spORF2 and H2O2, respectively. (C) Quantitative assessment of the average number of 53BP1 foci per cell (mean ± SD). Asterisks indicate significant difference determined by paired t-test with P = 0.003, 0.017 and 0.006 for L1, spORF2 and H2O2, respectively.
Figure 7.
Figure 7.
Full-length L1 and SpORF2 expression triggers senescence-like phenotype in normal human fibroblasts and human adult stem cells. (A) Normal human fibroblasts transfected with the empty vector, vectors expressing wt L1, or SpORF2, or treated with a DNA damaging agent zeocin. Blue color represents cells that express senescence-associated β-galactosidase. (B) Quantitation of the L1-induced senescence in normal human fibroblasts, BJ, immortalized with human telomerase. Black bars represent relative amount of senescent cells among human fibroblasts treated as described in (A). Grey bars represent relative amount of cells observed after each treatment in any given field. Asterisks indicate statistically significant difference between the cell numbers of cells transfected with the empty vector and cells transfected with the wt L1 expression cassette or zeocin treatment (t-test, P-value 0.014 and 0.00025, respectively). Untransfected (untr.) is mock-transfected cells. (C) Quantitation of the L1-induced senescence in human adult stem cells. Black and gray bars represent relative amount of senescent cells and total cells as described in (B). Asterisks indicate statistically significant difference between the cell numbers of cells transfected with the empty vector and cells transfected with the wt L1 expression cassette, zeocin treatment, or SpORF2 vectors (t-test, P-value 0.01, 0.0002 and 0.013, respectively).
Figure 8.
Figure 8.
A summary of the biologically relevant L1-related mRNA products and their respective impact on the host genome. Transcription of the functional L1 locus results in the production of either the full-length mRNA (FL1mRNA), the splice ORF2 mRNA (SpORF2mRNA) or both. FL1mRNA protein products can mobilize L1, Alu, and SVA elements, while SpORF2mRNA only produces ORF2 protein and as a result can only assist Alu retrotransposition. Expression of either L1 mRNA can generate ORF2, which leads to introduction of DNA DSBs potentially resulting in accumulation of mutations in the cellular genome.
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