Differential inhibition of LINE1 and LINE2 retrotransposition by vertebrate AID/APOBEC proteins
© Lindič et al.; licensee BioMed Central Ltd. 2013
Received: 4 August 2013
Accepted: 2 December 2013
Published: 17 December 2013
The role of AID/APOBEC proteins in the mammalian immune response against retroviruses and retrotransposons is well established. G to A hypermutations, the hallmark of their cytidine deaminase activity, are present in several mammalian retrotransposons. However, the role of AID/APOBEC proteins in non-mammalian retroelement restriction is not completely understood.
Here we provide the first evidence of anti-retroelement activity of a reptilian APOBEC protein. The green anole lizard A1 protein displayed potent DNA mutator activity and inhibited ex vivo retrotransposition of LINE1 and LINE2 ORF1 protein encoding elements, displaying a mechanism of action similar to that of the human A1 protein. In contrast, the human A3 proteins did not require ORF1 protein to inhibit LINE retrotransposition, suggesting a differential mechanism of anti-LINE action of A1 proteins, which emerged in amniotes, and A3 proteins, exclusive to placental mammals. In accordance, genomic analyses demonstrate differential G to A DNA editing of LINE retrotransposons in the lizard genome, which is also the first evidence for G to A DNA editing in non-mammalian genomes.
Our data suggest that vertebrate APOBEC proteins differentially inhibit the retrotransposition of LINE elements and that the anti-retroelement activity of APOBEC proteins predates mammals.
KeywordsG to A hypermutation AID APOBEC3 ORF1p Lizard APOBEC1 Zebrafish LINE2 LINE1 Zebrafish APOBEC2 Cytidine deaminase Retrotransposon
AID/APOBEC protein family members are cytidine deaminases (CDA) that convert cytidine to uridine in DNA and RNA . They emerged at the origin of vertebrates and have spread throughout the vertebrate lineage (summarized in Figure 1). The early ancestors of AID/APOBEC proteins have been identified in jawless and cartilaginous fish and include the activation-induced deaminase (AID), important for antibody diversification, and the APOBEC2 (A2) protein, involved in active demethylation and muscle development [35–38]. In amphibians, APOBEC4 (A4) and APOBEC5 (A5) proteins emerged, but their functions are yet to be identified [23, 24, 26]. During the tetrapod evolution, duplications of AID genes led to the development of APOBEC1 (A1) [24, 35, 39–41]. In placental mammals the APOBEC3 (A3) genes evolved and greatly expanded. While one gene is present in mice, there are 7 human A3 genes: A3A, A3B, A3C, A3D, A3F, A3G and A3H [24, 29–31, 35, 42, 43]. A3 proteins inhibit various viruses by introducing C to U mutations in viral cDNA intermediates leading to G to A hypermutations and viral inhibition (reviewed in ). Alternatively, A3 proteins may inhibit retroviruses and LINE elements by inhibiting reverse transcription [45–48]. A3 proteins have been shown to inhibit many LTR and non-LTR retrotransposons [49–54], but also various mammalian A1 and pre-mammalian AID proteins exhibit an activity against the human L1 (hL1) retrotransposon [55, 56]. Moreover, the effects of AID/APOBEC CDA activity have been identified in the sequence of several mammalian LTR and non-LTR retrotransposons [57, 58], but not yet in non-mammalian genomes, which lack A3 proteins. Recently, it has been shown that the lizard A1 protein possesses DNA deaminase activity , which is consistent with the hypothesis that the original function of AID/APOBEC family of enzymes is retroelement restriction, rather than antibody diversification .
To examine the hypothesis that retroelement restriction by APOBEC proteins predates mammals, we performed comparative analyses of the impact of currently available vertebrate APOBEC proteins on the retrotransposition of vertebrate LINE elements and performed genome-wide DNA editing analyses of vertebrate genomes. We found G to A DNA hypermutations in green anole lizard retrotransposons, which is the first evidence of retrotransposon editing in non-mammalian vertebrate genomes. The green anole lizard A1 protein displayed potent DNA mutator and anti-retroelement activity, which paralleled that of the human A1 protein. Our results further reveal a differential mechanism of action of A1 and A3 proteins against LINE retrotransposons.
Lizard A1 protein inhibits hL1 and ZfL2-1, but not ZfL2-2, retrotransposons
Zebrafish A2b and lizard A1 proteins exhibit DNA mutator activity
Human A3 proteins inhibit ZfL2-2 retrotransposition
Intact A3A active site is required for ZfL2-2 inhibition
The integrity of at least one A3B domain is required for ZfL2-2 inhibition
A3, but not A1 and A2, proteins decrease the amount of novel LINE DNA copies
Interestingly, despite their anti-retroelement activity against hL1 (Figure 3), the lizard and human A1 proteins did not change significantly the level of novel hL1 or ZfL2-2 DNA (Figure 8B). The possibility of A1 proteins affecting the level of the transfected plasmid or the level of expressed retrotransposon RNA was excluded by performing qPCR analyses (Additional file 1: Figures S5B and S6B). Our results thus suggest that the inhibition of hL1 retrotransposition by A1 proteins does not include significant alterations in the amount of novel retrotransposon DNA, thus implying that lizard and human A1 proteins do not interfere with the stability or the synthesis of the retrotransposon DNA.
Novel ZfL2-2 and hL1 DNA copies are not hypermutated
We next asked whether any mutations might have occurred in the novel spliced ZfL2-2 and hL1 DNA copies in the presence of zebrafish, lizard, mouse or human APOBEC proteins. The overall mutation rates were very low and there was no apparent increase in G to A mutations (Additional file 1: Figure S7), indicating that neither of the tested AID/APOBEC proteins edits the novel retrotransposon DNA during the course of reverse transcription or integration. This is in accordance with previous studies showing that the inhibition of hL1 by human AID/APOBEC proteins occurs in the absence of hypermutations [49, 51, 53–55, 66]. Although we cannot exclude the possibility of rapid degradation by host proteins of any highly mutated copies, or occurrence of mutations outside of the neo gene, our results suggest that the novel ZfL2-2 and hL1 DNA copies generated in the presence of the tested APOBEC proteins are not hypermutated.
Genome-wide analysis reveals G to A editing in lizard retrotransposons
Our genome analysis provides the first evidence of G to A editing of retrotransposon DNA in a non-mammalian vertebrate genome, thus reflecting the presence of editing competent AID/APOBEC proteins (or other deaminases). Importantly, we report for the first time a potent anti-retroelement activity of a reptilian A1 protein, which indicates that this host-defense activity of APOBEC proteins originates in amniotes. The DNA mutator and anti-retroelement activity of reptilian A1 proteins suggests that they indeed might have been responsible for this editing. Moreover, we demonstrate that human A3 proteins inhibit both L1 and L2 retrotransposons in a sequence-independent manner. In contrast, lizard and human A1 proteins blocked only ORF1p encoding LINE elements, revealing a differential mechanism of action of A1 and A3 proteins against LINE retrotransposition.
The anti-retroelement activity of A1 proteins predates mammals
AID/APOBEC proteins are present in all vertebrates (summary in Figure 1), but their distribution, evolution and functions differ. Fish genomes encode AID and A2 members of the APOBEC family, but lack the A1 and A3 genes . However, our genomic analyses did not reveal any DNA editing in zebrafish LINE elements (Figure 9). In accordance, our data demonstrate that zebrafish AID/APOBEC proteins have a negligible effect on ZfL2-1, ZfL2-2 and hL1 retrotransposition (Additional file 1: Figure S2). Along with the low DNA mutator activity of the zebrafish A2b protein, these results collectively indicate that the anti-LINE activity of APOBEC proteins evolved later during vertebrate evolution.
In reptiles and birds, A1 homologs have been discovered . Here, we confirm previous observations  that the anole lizard A1a protein is a potent DNA mutator (Figure 4) and show that it inhibits hL1 and ZfL2-1 retrotransposition (Figure 3), which suggests that the APOBEC anti-retroelement defense function exists in reptiles. The turnover of retrotransposons in reptiles is very rapid and only a few copies of each retrotransposon family are present in their genomes, suggesting that host factors efficiently prevent their fixation . The identification of the latter awaits further studies, but our results suggest that the lizard A1 protein could be one of the anti-retroelement proteins in reptiles. The lizard A1 protein is expressed in the gastrointestinal tract and it lacks the C-terminal region present in mammalian A1 proteins essential for their apoB RNA editing function [24, 41]. It has thus been suggested that the DNA editing function of A1 proteins predates their ability to modify RNA in mammals . Therefore, the DNA mutator and anti-retroelement activity of the lizard A1 protein, along with the G to A DNA hypermutations detected in the lizard genome, suggest that the anti-retroelement activity of A1 proteins emerged in amniotes.
A3 and A1 proteins differentially inhibit LINE retrotransposition
Cytidine deaminase activity and inhibitory potency of AID/APOBEC proteins against hL1, ZfL2-2 and ZfL2-1 retroelements
AID/APOBEC protein family
CDA in E. coli
Inhibition of retrotransposition
ND, edits viral cDNA 8
+ * / ++ 12
– * / +14
G to A DNA editing identified in a non-mammalian vertebrate genome
Whole genome analyses have previously provided evidence of DNA editing in LTR and non-LTR retrotransposons in human and mouse genomes . In this study, a clear G to A editing signal was identified in L1, but not in L2 retrotransposons of the lizard genome (Figure 9), despite their high diversity and low fixation rate [19, 20, 25]. These findings suggest that some retrotransposons are/were preferentially edited over others. Interestingly, both lizard and human A1 proteins interfered with hL1 and ZfL2-1, but not ZfL2-2, retrotransposons in vitro (Figure 3). On the other hand, the rates of editing observed in L1 and L2 retrotransposons in the vertebrate genomes analyzed were low. This is in accordance with the fact that despite the significant inhibition of retrotransposition (Figures 3 and 5), no lizard or human APOBEC-induced editing was detected in the novel hL1 DNA (Additional file 1: Figure S7), as shown previously for human APOBECs [49, 51, 53–55, 66], nor in ZfL2-2 DNA copies in our ex vivo assays (Additional file 1: Figure S7). Altogether, our results suggest that (1) G to A DNA editing of retrotransposons predates mammals, (2) that retrotransposons were differentially edited during evolution, and (3), that, based on the low editing rates of the vertebrate LINE retrotransposons and absence of hypermutations in the sequenced LINE copies, AID/APOBEC proteins may have impacted the evolutionary dynamics of LINE retrotransposons also in an editing-independent manner.
Altogether, the results of our ex vivo experiments, summarized in Table 1, along with our genomic analyses, indicate that the impact of AID/APOBEC proteins on the evolution of retrotransposons is broader than previously thought. The DNA editing detected in the lizard genome, the DNA mutator and the anti-retroelement activity of the lizard A1 protein shown here, suggest that the emergence of A1 proteins in reptiles is associated with the development of anti-retroelement activity in amniotes. Moreover, we observed that human and lizard A1 proteins restricted only ORF1p encoding LINE elements, while the presence of ORF1p was not necessary for anti-LINE activity of A3 proteins. Additionally, A3, but not A1, proteins potently restricted LINE retrotransposition by decreasing the level of novel retrotransposon DNA. The differential mechanism of LINE inhibition by A1 and A3 proteins, along with the differential editing of retroelements detected in vertebrate genomes, suggest that the early evolutionary appearance of APOBEC proteins may have importantly contributed to the different levels of expansion of LINE elements during evolution and enabled the vertebrates to cope with the harmful effects of retroelements.
Zebrafish AID, A2a and A2b encoding fragments were PCR amplified from previously described plasmids  using the primer pairs zAIDKpnIf/zAIDNotIr, zA2aKpnIf/zA2aNotIr and zA2bKpnIf/zA2bNotIr (Additional file 1: Table S1), and cloned into the pcDNA6.2 vector (Invitrogen). Lizard A1 variants F3 (R73K, Y116C) and F8 (N52D) coding fragments were amplified with lizA1HindIIIs and lizA1XhoIas primers from bacterial pTrc99A-based expression vectors that were a kind gift from prof. Conticello , and subcloned into the pcDNA6.2 plasmid. The AID and APOBEC bacterial expression plasmids used in the mutational assay were obtained by subcloning into the pTrc99A plasmid . Plasmids encoding the full length hL1 (pJM101 ), UnaL2 (pI-29 ), ZfL2-2 (pBZ2-5) and ZfL2-1 (p77.52)  retrotransposons, all of them marked with the neo cassette, were described previously. Plasmids encoding retrotranspositionally defective retrotransposons, the ZfL2-2 and ZfL2-1 3′ UTR deletion mutants (pBB4 and p74.18) , the hL1 ORF1p mutant (pJM111) and the ORF2p mutant (pJM105) with no reverse transcription activity , were used as negative controls. All retrotransposon plasmids also carried a hygromycin resistance gene that enabled selection of successfully transfected cells. Vectors expressing mouse A3 and human A1, A2, AID, A3C, A3F, A3G, A3D, A3H, A3H-L, A3A and A3B proteins were prepared from previously described plasmids [51, 78–81]. The plasmids encoding the A3A E72A, CC101,106AA, ECC72,101,106AAA, F75L and F95L mutants were prepared using the QuickChange II Site-Directed Mutagenesis kit (Stratagene) and the primers A3A.E72Af, A3A.E72Ar, A3A.CC101,106AAf and A3A.CC101,106AAr, as well as previously reported primers . NA3B and CA3B mutants encoding fragments were PCR amplified from the pKA3B plasmid [51, 61] using the primer pairs NA3B.HindIIIs/NA3B.XhoIas and CA3B.HindIIIs/CA3B.XhoIas, respectively, and cloned into the pcDNA6.2 plasmid. Plasmids encoding NA3B E68Q, A3B E68Q, A3B E255Q and A3B EE68, 255QQ mutants were prepared by site directed mutagenesis using the primers A3B.E68Qs, A3B.E68Qas, A3B.E255Qs and A3B.E255Qas. All plasmids were verified by sequencing. All primer sequences can be found in Additional file 1: Table S1.
E. colimutation assay
The mutation assay was performed as described previously [24, 63, 64] using the E. coli strain KL16 (Hfr (PO-45) relA1 spoT1 thi-1) and its UDG-deficient derivative (BW310). Briefly, KL16 and BW310 cells were transformed with AID/APOBEC expression constructs or vector alone. Individual colonies were picked and grown to saturation in a liquid LB medium containing 100 μg/ml ampicillin and 1 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG), with the exception of control experiments, in which the IPTG inducer was omitted. An aliquot of saturated culture was plated onto LB medium containing 100 μg/ml rifampicin and 100 μg/ml ampicillin to select RifR colonies, and an appropriate dilution was plated onto LB medium containing 100 μg/ml ampicillin for viable cell count. All plates were incubated overnight at 37°C to allow colony formation. Mutation frequencies were measured by determining the median number of colony forming cells that survived selection per 109 viable cells plated. Each median was determined from 8–16 independent cultures. IPTG induced expression of AID and APOBEC proteins conferred no obvious defect in cell growth or viability.
Human embryonic kidney 293T cells were transfected with 1 μg of APOBEC encoding plasmid. 48 h after transfection, cells were lysed in RIPA lysis buffer (1% NP-40, 0.1% SDS, 150 mM NaCl, 50 mM Tris–HCl, pH 7.6) with Protease Inhibitor Cocktail (Sigma) and incubated for 40 min at 4°C. After separation of cell lysates by SDS-PAGE, western blotting using α-HA (Sigma), α-Myc, α-V5 and α-GAPDH monoclonal antibodies (Invitrogen) and the appropriate HRP-conjugated secondary antibodies was performed. Detection with ECL reagent (Pierce) followed.
Retrotransposition and toxicity assays
The effect of AID/APOBEC proteins on retrotransposition and their possible cytotoxic effect were measured as previously described [51, 53, 60, 61]. Briefly, HeLa cells were seeded in 6-well dishes at a density of 2 × 105 cells per well, left to attach for 24 h, and co-transfected using 6 μl of Fugene® 6 Transfection Reagent (Roche), with 1 μg of a retrotransposon encoding plasmid (pJM101, pBZ2-5, or pI-29), or, for cytotoxicity testing, with the neo-expressing pcDNA3.1 plasmid (Invitrogen) instead, and with 1 μg of the respective AID/APOBEC protein encoding plasmid, or an empty vector (pcDNA6.2). In the case of the ZfL2-1 retrotransposon, a total of 1.6 × 106 cells per 10 cm plate were seeded and 24 h later co-transfected with 2.5 μg of each retrotransposon and AID/APOBEC encoding plasmid. 2 days post-transfection, cells were reseeded on 10 cm dishes, incubated for 24 h, and incubated with G418 (500 μg/ml) for the next 12 days. Resistant colonies were fixed with methanol, stained with trypan blue and counted. Assays with plasmids encoding the retrotranspositionally defective ZfL2-2 (pBB4) and ZfL2-1 (p74.18)  or hL1 (pJM111 and pJM105)  retrotransposons and plasmids encoding AID/APOBEC proteins were performed as negative controls. In addition, cells were transfected only with AID/APOBEC plasmids. In both cases, no resistant colonies were formed (data not shown). The retrotransposition rate in the presence of the empty pcDNA6.2 vector was set to 100%. Retrotransposition efficiency values are means ± SD from at least three independent experiments performed in triplicates. Statistical significance between two groups was calculated by performing the Student’s t-test. A value of P < 0.05 was considered to be of statistical significance.
DNA isolation with sequencing and qPCR analyses of novel LINE DNA and plasmid DNA levels
For sequencing and analysis of novel retrotransposon DNA HeLa cells were co-transfected with 1 μg of APOBEC and 1 μg of retrotransposon plasmids as described above, and transfectants containing the retrotransposon plasmid were selected with hygromycin (200 μg/ml) for 4 days. Total DNA (both chromosomal and episomal) was isolated using the DNeasy Blood & Tissue Kit (Qiagen) and contaminating RNA was degraded with RNase A from bovine pancreas (Roche).
To analyze the novel retrotransposon DNA for mutations (Additional file 1: Figure S7), 600 ng of DNA per sample in 20 μl reactions was subjected to Platinum Taq DNA Polymerase High Fidelity (Invitrogen) PCR amplification of the neo gene with primers neo437S and neo1808AS . The integrity of the isolated DNA was verified by amplification of the single copy PGK gene. After gel electrophoresis, the intronless neo PCR products were isolated, purified and cloned into pCR4 TOPO TA cloning vectors (Invitrogen). Plasmids were isolated from at least 5 individual colonies and the neo insert was sequenced using T7 primers. Nucleotide sequences of independent clones were aligned and analyzed using the Hypermut software .
To determine the relative abundance of novel ZfL2-2 and hL1 copies in total DNA, quantitative PCR analysis was performed. A 117 bp fragment of the spliced and reversely transcribed neo gene was amplified with an internal sense primer (Neo210s) and an exon-exon junction spanning antisense primer (Neo1228as), which specifically recognizes the spliced neo cassette. Primers targeting two single-copy genes, superoxide dismutase (SOD2) and haemoglobin beta (HBB) , were used for normalization of spliced neo DNA levels. All reaction mixtures contained 500 nM of each primer and 2.5 ng of DNA in a final volume of 10 μl. qPCR was carried out on a LightCycler Nano system (Roche) using FastStart Essential DNA Green Master (Roche) chemistry at the following cycle conditions: one cycle at 95°C for 600 s; 45 cycles at 95°C for 15 s, 60°C for 12 s, 72°C for 15 s, and one cycle at 72°C for 120 s. The specificity of each primer pair was confirmed by melting curve analysis and gel electrophoresis following each run (Additional file 1: Figure S8). A calibrator DNA sample, isolated from HeLa cells co-transfected with the retrotransposon and an empty plasmid was used for determination of PCR reaction efficiency and for inter-run variability correction. An established comparative Cq method with efficiency correction, error propagation and calibrator normalization was used for relative quantification using both reference genes . Results are presented relative to the neo frequency obtained with the DNA sample isolated from cells co-transfected with an empty vector and the retrotransposon plasmid.
To test the effect of APOBEC proteins on plasmid DNA, cells were co-transfected as described above, either with the ZfL2-2 encoding plasmid or an empty pcDNA6.2 vector, and the APOBEC encoding plasmid. 48 h after transfection, total DNA was isolated, and the effect of APOBEC proteins on plasmid DNA was analyzed with qPCR. Levels of the ZfL2-2 retrotransposon encoding plasmid were measured by amplification of a 109 bp fragment using the primer pair Neo673F/Int738R, while pcDNA6.2 plasmid level was detected with the primer pair Blas149F/Blas248R amplifying a 100 bp long fragment within the blasticidin resistance gene. Plasmid levels were normalized to genomic DNA levels by measuring the HBB and SOD2 gene levels, and the reaction conditions were essentially the same as described above, except that 0.025 ng of total DNA was used per reaction. All primer sequences are listed in Additional file 1: Table S1.
RNA isolation, reverse transcription and qPCR analysis
RNA was isolated 2 days post-transfection using the RNeasy Plus Mini kit (Qiagen) and contaminating DNA was degraded using the DNA-free Kit (Ambion). Reverse transcription was performed in 10 μl reactions with 1.25 μg of each RNA sample using the Superscript Vilo cDNA Synthesis Kit (Invitrogen). Negative controls without template or reverse transcriptase were included in all assays. Resulting cDNA was diluted 4000-fold and subjected to qPCR analyses with primer pair Neo673F/Int738R (Additional file 1: Table S1). The GAPDH reference gene was amplified with the GAPDHf/GAPDHr primer pair. The reaction conditions were the same as for the DNA qPCR analysis described above.
Genome-wide analysis of G to A editing in vertebrate genomes
All genomic repeats in the LINE and LTR classes were downloaded from the UCSC table browser  ‘rmsk’ table via the “Galaxy” online genomics tool (http://main.g2.bx.psu.edu/) [86–88] for mouse, lizard and zebrafish genomes (mm9, anoCar2 and danRer7 genomic builds, respectively). Additionally, we downloaded the latter 2 genomes from the UCSC genome browser’s  FTP site (ftp://hgdownload.cse.ucsc.edu/) and ran RepeatMasker (http://www.repeatmasker.org) with libraries of repeats of interest that were not present in the ‘rmsk’ table (see Additional file 2). The genomic sequences respective to the coordinates identified by RepeatMasker were extracted from the genomes and united with the ‘rmsk’ table data. Retrotransposon sequences of each genome were sorted by subfamilies. The subfamily annotation was assigned by RepeatMasker and was derived from Repbase Update’s  three levels of classification that we addressed as ‘Class’ , ‘Family’ and ‘Subfamily’ (e.g. LINE class, L1 family, L1_AC_11 subfamily (Additional file 1: Tables S2 and S3)). The DNA editing detection was performed using the method described by Carmi et al. . The method contains two steps: generating pairwise alignments of all sequences of the same subfamily using BLAST  and screening the alignments for DNA editing with PERL scripts. Briefly, pairwise alignments between similar sequences in LINE and LTR classes of retrotransposons from the zebrafish, lizard and mouse genomes were generated using BLAST . The alignments were then screened for one or more clusters of consecutive G to A mutations, and the resulting editing rates compared to those previously detected in the human (L1 and SVA) and mouse genomes (L1 and IAP) . Based on the strand specificity of DNA editing, complementary event of C to T mutations was used as control. The goal was to detect pairs of ‘parent’ and ‘child’ sequences, where the child has a cluster of G to A mutations in comparison to the parent, denoting that its DNA was edited by APOBEC proteins during retrotransposition. The high similarity we demanded between these two sequences, enforced by the BLAST parameters (E-value of 10-50) and our algorithm, provides significant support that the child sequence indeed evolved from the parent sequence. The DNA editing finding algorithm searches for a pair of sequences with a series, or cluster, of G to A mismatches. To reduce the false-positive rate, we do not allow any other type of mismatch in the G to A cluster. The cluster’s significance is measured by two values: (i) ‘Threshold’ - the number of G to A mismatches in the cluster, (ii) ‘P-value’ - the P-value calculated by the Binomial probability of arbitrarily getting such a cluster of transitions in the given alignment. The probability of a single transition is calculated by the number of T to C and C to T transitions in the alignment. A3 proteins introduce G to A mutations in the retrotransposon’s positive strand as defined by its ORF. For control, we exploited this strand specificity by using the G to A ‘editing’ of the minus strand to test the significance of our results. Our data contains only the retrotransposon positive strands, thus, this event is represented as C to T ‘editing’ in our data. We present the same parameters used by Carmi et al. , which contain a set of parameters with extremely high significance (P-value = 1e-13 and Threshold = 9) and a set of slightly relaxed parameters (P-value = 1e-8 and Threshold = 8). The data in the manuscript was generated using the latter (for details, see also Additional files 1 and 2). We applied the method to all LINE and LTR repeats in the zebrafish and lizard genomes and compared the results to significantly edited families in human and mouse genomes.
Long terminal repeat
Long interspersed nuclear element
- ORF1p and ORF2p:
Open reading frame 1 and 2 retrotransposon proteins
Cytidine deaminase activity
Neomycin resistance gene
We thank Shai Carmi for his help at the early stage of the project, and Norihiro Okada, John Moran, Bryan Cullen, Boris Matija Peterlin, Rai Kunal, Silvestro Conticello and Yong-Hui Zheng for reagents. We thank Alenka Čopič for critical reading of the manuscript. This work was supported by the Slovenian Research Agency [31952 to NLi, J7-2230, J1-2141 to NLo and MB, J3-4168 to MB and P1-0207 to NLo and TP]. BAK and EYL were supported by the Israel Science foundation [728/10] and by the European Research Council . Funding for open access charge: 31952 to NLi.
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