LINE-1 ORF1 protein enhances Alu SINE retrotransposition

Abstract

Retroelements have contributed over one third of the human genome mass. The currently active LINE-1 (L1) codes for two proteins (ORF1p and ORF2p), both strictly required for retrotransposition. In contrast, the non-coding parasitic SINE (Alu) only appears to need the L1 ORF2p for its own amplification. This requirement was previously determined using a tissue culture assay system in human cells (HeLa). Because HeLa are likely to express functional L1 proteins, it is possible that low levels of endogenous ORF1p are necessary for the observed tagged Alu mobilization. By individually expressing ORF1 and ORF2 proteins from both human (L1RP and LRE3) and rodent (L1A102 and L1spa) L1 sources, we demonstrate that increasing amounts of ORF1 expressing vector enhances tagged Alu mobilization in HeLa cells. In addition, using chicken fibroblast cells as an alternate cell culture source, we confirmed that ORF1p is not strictly required for Alu mobilization in our assay. Supporting our observations in HeLa cells, we find that tagged Alu retrotransposition is improved by supplementation of ORF1p in the cultured chicken cells. We postulate that L1 ORF1p plays either a direct or indirect role in enhancing the interaction between the Alu RNA and the required factors needed for its retrotransposition.

Figure 1. Schematic of the LINE and SINE assay

RNA transcription is performed by L1 5′UTR promoter or the internal pol III promoter of the SINE enhanced by the 7SL upstream sequence. An intron (a self-splicing in the SINE vector) interrupts the neomycin (neo) resistance gene (hatched box) and promoter present in an inverted orientation. Because of orientation, the intron will splice out only from the transcripts generated by the retroelement’s promoter. The RNA is reverse transcribed, followed by integration of the cDNA into the genome. The new insert contains a functional neomycin gene.

Figure 2. L1 ORF1p enhances retrotransposition of a tagged Alu but not L1

A. L1 ORF1p gradient and retrotransposition activity of L1 and Alu (driven by ORF2_RP) in HeLa cells. Cells were transfected with L1-tag (open circle) or Aluneo^TET plus ORF2 (black circle) and supplemented with increasing concentrations of ORF1. The mean G418^R colonies for each retroelement are plotted as a solid line, where error bars represent the standard deviation and the flanking discontinuous lines represent the 95% confidence interval. The calculated slope for Alu is 95.12 ± 11.92 and for L1 -12.70 ± 3.55 and shown to be significantly different as determined by Least squares regression analysis, p = 0.00013 n = 3. B. Alu-tag RNA transcription and processing is unaffected by the supplementation of ORF1p. Representative northern blot analysis of polyA selected RNA from transfected cells with the tagged Alu vector alone (C, control), or supplemented with ORF2p plus different amounts of ORF1p (0 – 4 μg). The unspliced (open arrowhead), spliced RNA (small arrow) and actin bands are indicated. Four separate transfections and northern blot analyses were performed for the quantitative evaluation. The numbers below represent the relative mean ± SD of Alu-tag spliced (β-actin corrected) relative to the control (C) which was arbitrarily defined as “1”. No significant differences between the amounts of the spliced Alu-tag RNA of any of the experimental conditions relative to the control (Student paired t-test p ≥ 0.23).

Figure 3. Both human and rodent ORF1p enhance ORF2 driven Alu retrotransposition

HeLa cells were transfected with the tagged Alu supplemented with an ORF2p expression vector from the human L1_LRE element or the rodent L1_A102 or L1_spa elements (n = 5). Each Alu-ORF2p set was cotransfected with different amounts of the corresponding human/rodent ORF1p expression vector or empty vector for the “0 μg” or ORF2 only control. The relative G418R colonies were graphed using the ORF2 only control (black column) reference, which was arbitrarily assigned as 100. The p-values (Student paired t-test) for significant differences are indicated above the columns.

Figure 4. A ORF2p is sufficient to mobilize SINEs in chicken cells

A. Retrotransposition activity of tagged Alu supplemented with the synthetic ORF2s from the human L1_RP or the mouse L1_spa in chicken embryo fibroblast cells. The mean of the total number of G418 resistant colonies for each time point is shown (n= 5). No colonies were ever observed for the Alu-tag cotransfected with the empty vector (control) indicated by “0”. B. Evaluation of the Alu inserts from the chicken cells. Alu inserts were evaluated by PCR analysis using primers to the Alu sequence and the neomycin gene sequence (shown as bold arrows in Figure 1). DNA was recovered from pooled G418 resistance colonies generated in the chicken embryo fibroblast cells transfected with the tagged Alu vector plus the synthetic versions of either human (L1_RP) or mouse (L1_spa) ORF2 with and without supplementation of the corresponding ORF1p. An open arrowhead indicates the PCR product corresponding to an insert containing the spliced version (open arrowhead) of the Alu expression vector. The Alu expression plasmid (P lane) was used as the control for unspliced products (small arrow). DNA from untransfected cells were used as the negative control (−). M is DNA marker. C. ORF1p enhances SINE retrotransposition in chicken cells. Cells were transfected with the tagged Alu plus the human ORF2p (L1_RP) supplemented with different amounts of the ORF1p expression vector (0, 1, 2 or 6 μg). Columns represent the mean number of G418 resistant colonies for each time point (n = 5) and S.D. shown as error bars. Results significantly different from the no ORF1 reference transfection with p-values of p≤ 0.05 (Student paired t-test) are indicated by an asterisk (*).

Figure 5. Model of potential roles of ORF1p in SINE retrotransposition

Although onlyORF2p is strictly required for SINE retrotransposition, supplementation with ORF1p enhances their mobilization. There are two potential scenarios of how ORF1p increases Alu retrotransposition. First, because of the chaperone nature of ORF1p, the direct interaction of ORF1p with the SINE RNA may play a role in protecting the transcript from degradation or aiding the RNP complex to reach the nucleus. Alternatively, in the second scenario, ORF1p may have an indirect role by interacting with other components such as ORF2p or cellular factors to facilitate retrotransposition. In addition, ORF1p could have a role in increasing the half life of ORF2p or targeting other cellular factors and ORF2p to the nucleus.