Identifying related L1 retrotransposons by analyzing 3' transduced sequences
© Szak et al.; licensee BioMed Central Ltd. 2003
Received: 9 December 2002
Accepted: 24 March 2003
Published: 16 April 2003
A large fraction of the human genome is attributable to L1 retrotransposon sequences. Not only do L1s themselves make up a significant portion of the genome, but L1-encoded proteins are thought to be responsible for the transposition of other repetitive elements and processed pseudogenes. In addition, L1s can mobilize non-L1, 3'-flanking DNA in a process called 3' transduction. Using computational methods, we collected DNA sequences from the human genome for which we have high confidence of their mobilization through L1-mediated 3' transduction.
The precursors of L1s with transduced sequence can often be identified, allowing us to reconstruct L1 element families in which a single parent L1 element begot many progeny L1s. Of the L1s exhibiting a sequence structure consistent with 3' transduction (L1 with transduction-derived sequence, L1-TD), the vast majority were located in duplicated regions of the genome and thus did not necessarily represent unique insertion events. Of the remaining L1-TDs, some lack a clear polyadenylation signal, but the alignment between the parent-progeny sequences nevertheless ends in an A-rich tract of DNA.
Sequence data suggest that during the integration into the genome of RNA representing an L1-TD, reverse transcription may be primed internally at A-rich sequences that lie downstream of the L1 3' untranslated region. The occurrence of L1-mediated transduction in the human genome may be less frequent than previously thought, and an accurate estimate is confounded by the frequent occurrence of segmental genomic duplications.
Analysis of the initial draft of the human genome revealed that 45% of the sequence is transposable elements . The expansion of the human genome that resulted from the mobilization of these transposable elements suggests they hold secrets of our evolution and increase the plasticity and variation in our genome. In some cases, transposable elements may have been domesticated by their host to serve clear functional roles [2–10]. Most human transposable elements are retrotransposons.
Among the retrotransposons in the human genome is the LINE-1 (L1) element. A full-length L1 insertion in the genome is approximately 6,000 nucleotides long and consists of a 5' untranslated region (UTR), two open reading frames (ORFs), and a 3' UTR terminating in a poly(A) tail . The second ORF of L1 encodes three domains critical for L1 propagation: endonuclease (EN) , reverse transcriptase (RT) [13, 14], and a 3' terminal zinc-finger-like domain . The EN and RT nick a target site in DNA and reverse transcribe L1 RNA, respectively, to integrate into a new genomic locus [12, 16–18]; this process is known as target-site-primed reverse transcription (TPRT). It is believed that the tendency of EN to nick target DNA at the consensus 3'-AA-TTTT-5' exposes a T-rich sequence, to which the poly(A) tail of an L1 transcripts can anneal thereby priming reverse transcription [16, 19–21]. A new L1 insertion is usually flanked by short direct repeats derived from the target DNA locus upon L1 integration [22, 23]; these repeats are referred to as target-site duplications (TSDs).
The role of L1 in shaping the human genome is unmistakable. Not only does L1 sequence itself contribute at least 462 megabases (Mb) to our genome (17% of the total length) , but copies of the Alu and SVA transposable elements and processed pseudogenes are also believed to have inserted into the genome by borrowing the EN and RT proteins encoded by L1 [13, 20, 21, 24–27]. In addition to self-mobilization and mobilization of other transposable elements, L1s can also move unique flanking DNA sequence to another locus in the genome in a process known as 3' transduction. This occurs when an L1 transcript reads into a portion of the downstream flanking sequence. This 3' sequence becomes transduced, along with the L1 sequence, to a new genomic locus; a hypothesized cause of the imprecision of the 3' end of the L1 transcript is the weak polyadenylation signal in the L1 element . Clear indications of 3' transduction have been documented in cases where an L1 inserted into the dystrophin gene , APC  and CYBB . All these disease-producing L1 insertions, the boundaries of which were defined by flanking TSDs, contained novel sequences downstream of the L1 sequence itself. In addition, it has been suggested that the multiple copies of exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene found in the human genome may have proliferated via L1-mediated transduction . In most of these cases, the progenitor L1 element could be identified on the basis of the sequence of the 3'-transduced DNA segment.
A proposed consequence of L1 3' transduction is exon shuffling [28, 33, 34]. That is, an exon downstream of an L1 may be co-mobilized with that same L1 and inserted at a new locus such that the exon is integrated into another gene. Moran et al. demonstrated this experimentally in cultured cells by cloning a reporter gene containing a splice acceptor downstream from the polyadenylation signal of an intact L1 element . This engineered L1 retrotransposed into transcriptionally active genomic loci, allowing the co-mobilized reporter to be expressed after being spliced into a transcript expressed in these cells, effectively creating a chimeric mRNA .
We have previously found that nearly 9% of recent L1 insertions in the human genome have TSDs that are consistent with 3' transduction . That is, the 3' TSD of these L1s with transduction-derived sequence (L1-TDs) is preceded by a poly(A) tail and located up to several hundred nucleotides downstream from the end of the L1 3' UTR [35, 36]. On the other hand, standard L1 insertions have TSDs that follow a poly(A) tail immediately flanking the L1 sequence. For L1 elements that have 3'-transduced sequence, sibling, progenitor, and/or descendant L1s can be identified by comparing the transduced sequence to the sequence downstream of other L1 elements in the genome.
Using a recently developed algorithm, TSDfinder [23, 37, 38], we have precisely identified L1 insertions in the human genome whose sequence signature suggests an L1-TD. We then determined which of these transduced sequences shared high similarity with one or more other genomic loci that were also located immediately downstream of an L1. In this way, we built families of L1s potentially derived from the same progenitor element. We found that many potential family members of L1-TDs were merely duplications. Bona fide transduced sequences were analyzed for functional annotation, such as coding regions of genes, in the human genome. In studying the architecture of the 3'-transduced sequences, we found that only a fraction had a recognizable polyadenylation signal. For some of the other transduced sequences, in lieu of a polyadenylation signal, the pairwise alignment between the presumed progenitor element plus its downstream sequence and a descendant L1-TD ended in poly(A) or a related A-rich sequence. These sequence structures may indicate internal priming of the L1 RT at A-rich tracts of a transcript during the process of TPRT.
We used the RepeatMasker  and TSDfinder [23, 37, 38] programs to identify 6,178 L1 elements that had a sequence structure consistent with 3' transduction in a recent build of the human genome. These L1s had TSDs at least nine nucleotides long preceded by poly(A); they were classified as L1-TDs on the basis of having at least 20 nucleotides of sequence between the end of the 3' UTR and the start of the poly(A) tail that immediately preceded the 3' TSD. These L1-TDs represented 38% of all L1s for which we were able to identify TSDs .
Identifying related L1s
For these 3'-transduced sequences to be legitimate, they had to be located downstream of another L1 elsewhere in the genome. Otherwise, the mechanism for their mobilization or duplication in the genome might not be L1-dependent. To test for this, we collected 3 kilobases (kb) of sequence downstream from each 3' intact L1 that we found in the genome , formatted this collection of 3'-flanking sequences as a BLAST database , and queried each putative transduced sequence against it (see Materials and methods).
Overview of L1-TD based families
L1 id (Chrom_number)
L1 5' start in Gi*
L1 end or end of transduced sequence in Gi*
Length of transduced sequence (nucleotides)
L1 length (nucleotides)
Families of L1 elements
For each family of L1 elements, we tried to determine the relationship between the family members. That is, an L1-TD is either a sibling of the other family members found, or it is the child of a progenitor element. In addition, an L1-TD could be the progenitor of subsequent L1-TDs, giving rise to composite transduction events. For an L1 to be a bona fide progenitor element of a L1-TD, it must be longer than, or of the same length as, the L1 element of the L1-TD. Furthermore, in order to have been transcribed and transposed to give rise to progeny elements, the progenitor L1 must in principle be long enough to include the internal promoter in the 5' UTR. The majority of L1s in the genome are 5' truncated , and over time, a full-length L1 may be disrupted by mutation, insertion of another transposable element, or other DNA rearrangements. Consequently, in our set of 25 families, only 10 have a nearly full-length candidate for the progenitor element; their family ids are: 5, 6, 7, 11, 12, 14, 20, 21, 23, and 25 (Table 1). For the remainder of the families, although downstream sequences were similar and TSDs marked the end of aligned sequences, the L1s in a given family were either shorter than the L1 for which a transduced sequence was found, or too short to have been transcribed from the internal L1 promoter. This would imply that, in many of our cases, L1s in a family are siblings that arose from the same progenitor L1.
Four of the L1-TDs in our final set appeared to be composite transpositions. That is, we identified an L1 with downstream sequence that matched the proximal part of the transduced sequence, but we did not find any sequence downstream from an L1 that matched the distal end of the transduced sequence.
Functional annotation of the transduced sequences
We next studied the location of all the transduced sequences in Table 1 on the set of annotated 'NT_' contigs assembled at the National Center for Biotechnology Information (NCBI). In particular, we were interested to see if any transduced sequences were annotated as an exon, lending direct support to the mechanism of L1-mediated exon shuffling [28, 33, 34]. None of the transduced sequences downstream of any of the L1 family members in Table 1 was annotated as an exonic sequence. Of the 63 sequences that make up these families 12 were annotated as intronic sequences. One of the sequences was within 1,650 nucleotides of the start of an mRNA annotation predicted by automated computational analysis (L1 id 13_10012 and gene LOC92404 that is similar to a putative protein-tyrosine phosphatase). Thus, it is possible that this transduced sequence contributes important regulatory elements to the promoter region, influencing the expression of this gene. However, it is important to note that because our studies are confined to relatively young elements with TSDs, our failure to identify such examples by no means rules out exon shuffling by 3' transduction as a potentially important evolutionary mechanism. For example, one such event appears to have occurred 7-10 million years ago (Mya) with exon 9 of CFTR, the caveat being that there is no L1 element upstream of this particular exon in CFTR itself . In our analysis, we found three transduced sequences with similarity to CFTR exon 9; however, two of them were part of a segmental duplication, and the third had a nearby sequencing gap, precluding assessment of its duplication status.
Poly(A) tails of L1s
L1 id (Chrom_ number)
Alignment ends in A-rich stretch*,†
For five of the transduced sequences lacking a clear polyadenylation signal, the alignment between the 3'-transduced sequence and family members ended at an A-rich sequence; the 3' TSD of the 3'-transduction event was found immediately downstream of this A-rich sequence in the DNA (Figure 3b). A similar sequence structure was reported by Ovchinnikov et al. for a 3'-transduction event . Several possible explanations for this sequence structure are addressed in the Discussion. One explanation given by Ovchinnikov et al.  is that the transcripts may have been internally primed at an A-rich sequence (see Additional data file and ); a different type of internal priming is also required by a current model for 5' inversion of L1s .
It is possible that the L1 sequence has evolved to avoid long internal tracts of As in order to prevent internal priming of the transcript. The L1 consensus sequence has a 40% A content, not including the 5' UTR. We calculated the probability of finding an eight-nucleotide-long tract of As in a DNA sequence with such a sequence composition is 87%. However, the longest stretch of As found in the L1.3 reference sequence is seven (probability of 99%), and there are four such tracts.
Finally, we observed that for two of the L1-TDs that do not have a polyadenylation signal, the sequence that the TSDfinder program defined as the poly(A) tail is 'patterned'; that is, the tail is composed of A-rich simple repeats (for example, AAATAAAT...) . Since these poly(A) tails themselves contain polyadenylation signals, it is possible that their assignments as poly(A) tails are false positives, and the real tail formed by polyadenylation has shortened over time and is not detectable [44, 46, 47]. Four of the remaining L1-TDs for which no polyadenylation signal was found appeared to result from multiple sequential transduction events; we did not have family members that align with the 3' end of the transduced sequence to determine whether the poly(A) stretch could have been encoded in DNA.
In this study, we used computational methods to analyze human L1s whose sequence structure was consistent with 3' transduction. The vast majority of these putative L1-TDs could not be thoroughly evaluated for family members because of gaps in the genome sequence, excessive repetitive DNA content, sequence duplications and other practical limitations. Therefore, we are unable to calculate the overall rate of transduction events. Of the putative 3' transduced sequences, 58% (3,562) lacked detectable BLAST hits in our database of sequences downstream from L1s. Possible reasons for this are: first, that the sequence is unique in the human genome and was never 3' transduced (false positive); second, that the sequence has a counterpart in the unsequenced portion of the human genome; third, the loss of the full-length progenitor L1 due to recombination , or fourth, that the progenitor L1 sequence suffered from extensive mutations that precluded its detection as a 3' intact L1, and therefore, the downstream sequence was not included in our BLAST database.
It is surprising how few of the examples of putative L1-TDs can be directly verified by finding either progenitor-progeny or sibling pairs. This can be understood by considering the facts that the human population is highly outbred, and L1 elements are preferentially cis-acting [24, 27]. Progenitor elements for L1-TDs must be transpositionally competent and thus are likely to be relatively young. Their youth means that they are likely to be present in the human population in an 'unfixed' (heterozygous) state and in a relatively small population. Once such an element spawns a progeny element, the progenitor element (as well as the progeny element) has a high likelihood of extinction due to genetic drift; the donor and the progeny elements will be separated from each other by outcrossing (see  for further discussion). Alternatively, Boissinot et al. have hypothesized that full-length L1 elements may be selectively removed from the genome by recombination and thus not be found .
We were unable to build families around a high proportion of our initial L1-TDs because of their residence in duplicated genomic regions. This finding is consistent with data showing an abundance of both interchromosomal and intrachromosomal duplications in the human genome . It is unclear how many of these duplications are due to errors in genome assembly and how many represent authentic segmental duplications; correctly assembling duplications as a genuine landscape features of the genome sequence is a formidable informatics challenge . In the future, as better, more accurate, genome builds become available, particularly with regard to the presence of duplications and other rearrangements and the annotation of genes and their promoter regions becomes more thorough and correct, it will be important to repeat this study for the whole genome sequence.
Although in a relatively recent genome build we did not find any clear examples of transduced exons, we did find one transduced sequence located less than 2 kb upstream from a putative gene. This particular transduced sequence could contribute to the regulatory regions of that gene. To find examples of exons shuffled via L1 retrotransposition, an analysis similar to the method we used in this study may have to be performed with older subfamilies of L1s. According to RepeatMasker annotation, all the L1-TDs in Table 1 are of a primate-specific lineage, and it is believed that the ancestral primate genome, the structure of which is thought to be very similar to the modern human genome, existed 60-70 Mya . Thus, at the earliest point in time that we can reliably detect intact L1 elements, most human genes may have been largely established. That is, older L1s and older families of LINEs may have had more influence on the exon composition of genes which themselves are generally rather old. Nevertheless, until more complete genome sequences are available for comparative genomic analyses, we can only speculate on the mechanism by which genes have been altered, sometimes contributing to the formation of a new species. Furthermore, although GC- and gene-rich regions generally lack L1s , it has been hypothesized that at any given time during the evolution of the human genome, L1s have inserted randomly, but over time, they have been selected against in GC-rich regions . If this is true, it may be difficult to find evidence in our genome of L1-mediated exon shuffling.
Some L1s with 3'-transduced sequence lack a polyadenylation signal but a common stretch of As delineate the end of an alignment between family members (Figure 3b). One explanation for this sequence structure is that the transcripts were polyadenylated at the same location, and the polyadenylation signal mutated beyond recognition after the insertion event occurred; interestingly, Ovchinnikov et al. reported that polyadenylation signals tend to degrade rapidly after L1 insertion . A second explanation is that the poly(A) sequence is encoded in DNA and either the transcript was degraded up to the position of the A-rich sequence or RNA polymerase II failed to elongate the message past the A-rich sequence; the resulting transcript would then resemble one that had been polyadenylated. Alternatively, there may be a propensity for the L1 transcript to break at A-rich sequences, resulting in an A-rich 3' end that would mimic a 3' polyadenylated transcript.
Interestingly, a recently proposed mechanism of 5' inversion of L1 elements requires that first-strand cDNA synthesis be primed internally on the L1 transcript . Therefore, it is possible that the cDNA synthesis of a 3'-transducing transcript may also be primed at an internal A-rich site as suggested by Ovchinnikov et al.  (see Additional data file). Since the L1 EN consensus site is 3'-AA-TTTT-5', the first cut would expose a T-rich sequence on the target sequence from which an A-rich template could be primed for reverse transcription. Indeed, the TSDs for the 3'-transduction events that lack a polyadenylation signal are A-rich at their 5' ends (Table 1), indicating that the target site at which priming occurred would have been T-rich.
We did not find evidence of internal priming for standard L1 insertions predicted to produce 3' truncated L1 elements with well defined endpoints. Rather, L1 elements that are 3' truncated were mostly interrupted by insertions of another transposable element. Lack of internal priming on a standard L1 transcript may be due to interference by the L1 ORF1 protein that has been reported to bind specifically to L1 RNA . Moreover, it is possible that the L1 transcript has a secondary structure that would inhibit an A-rich region from being available as a template for reverse transcription, whereas a 3' tail on the L1 transcript, especially if derived from transduced sequence, may be much more accessible. Finally, in the L1 sequence minus the 5' UTR, the length of the longest internal A-tract (seven nucleotides) is shorter than would be expected by random chance. Thus L1 may have evolved multiple mechanisms to avoid internal priming on A-rich tracts, which would generate defective elements, while allowing it to occur in the flanking sequences, where polyadenylation signals might or might not be found.
Materials and methods
The human genomic DNA sequence records used were the set of contigs assembled at the NCBI  as of 29 August 2001 (build 25). L1 elements in the contigs were annotated using RepeatMasker  with a custom library containing only the L1.3 element (GenBank accession number L19088.1); in this way, we dealt primarily with young L1 elements . The TSDfinder program [23, 37, 38] was run on the RepeatMasker *.out output files in order to find the TSDs of the L1 elements, thereby refining the L1 boundaries. Each time a 3' intact L1 insertion was found (no more than 30 nucleotides missing from the 3' end of the 3' UTR), the 3 kb of sequence downstream from the L1 was collected. A total of 72,582 such 3-kb sequences were collected. These sequences were placed into a file and formatted as a BLAST database (formatdb parameters: -n ThrPrSeqDB -p F -V T -o T).
During the same run of the TSDfinder program, L1-TDs were identified. To be classified as an L1-TD, the distance between the end of the L1 3' UTR and the start of the 3' TSD had to be greater than 20 nucleotides (not counting the length of the poly(A) tail preceding the TSD nor the number of nucleotides missing from the end of the L1 3' UTR according to the RepeatMasker annotation of the L1 element). The classification of an L1 insertion as an L1-TD was only allowed when a 3' TSD closer to the L1, indicating a standard insertion event, could not be found (see [23, 37, 38]). The coordinates of the candidate transduced regions (the gi record and the begin and end coordinates) were stored for later analysis.
Analyzing putative 3'-transduced sequences
Transduced sequences were masked to avoid multiple ambiguous matches. The masking was accomplished using the default settings of RepeatMasker  (parameter -xsmall). For 2,085 (33%) of the 6,178 L1-TDs, the putative transduced sequence was nearly completely masked. The blastn program  was run for the putative transduced sequences against ThrPrSeqDB (parameters: -d ThrPrSeqDB -e 0.05 -J T -U T -F 'm D;R' -Z 150). By doing so, we ensured that any significant match in the genome was also downstream of a potential progenitor L1 for any particular transduced sequence.
A series of Perl scripts was used to examine the BLAST results (see rationale in Figure 1). To test for duplications, the 3 kb of downstream sequence for each putative L1-TD and the potentially related L1(s) identified by BLAST were collected. These sequences were input into bl2seq  (parameters: -g T -F 'm D;R' -S 1 -e 10 -X 100 -q -1). If the alignment between the sequences extended beyond the end of the putative transduced sequence, the sequence was labeled as a duplication and was not analyzed any further. Some sequences were removed from analysis because of nearby sequencing gaps that precluded a conclusion regarding the duplication status. Finally, for some alignments produced by bl2seq, the alignment was less than 90% identical or was considered too poor an alignment to continue further analysis with that particular set of L1s.
The duplication status could not be properly assessed for 154 of the initial putative transduced sequences, and they were consequently removed from consideration. One reason for ambiguity of the duplication status was gaps in the genome sequence; if either the query or the subject L1 had a stretch of more than 50 Ns in the 3 kb of downstream flanking sequence, indicating a gap in genome sequence, these were excluded from the analysis because they tended to interfere with the assessment of duplication and confound the automatic analysis. No proof of mapping to a genomic duplication was detected for 93 of the 6,178 initial 3' transduction candidates and their respective family members. These 93 families were made up of 652 total members. The DNA sequence of each member of these families was collected and multiple alignments among the family members were performed using the clustalx and GeneDoc software [53–55]. Manual inspection of the alignment of the family member sequences revealed that 12 of these families had more than 10 members, and it was immediately clear that the matches with the putative transduced sequences in these families were based solely on patchy alignments of largely low-complexity sequence. For the remaining 81 families, 43 were eliminated because of low-complexity matches only (largely poly(A) sequence) or previously missed duplications in the 3' flank. One of the families was eliminated because both L1 elements were full length, yet one of them had a 131-nucleotide insertion in its 5' UTR and the other did not, indicating that these L1s were not directly related [23, 56]. Finally, for nine families, although alignment of 3'-transduced sequence with a family member was clearly delineated by the 3' TSD, we found sequence duplication in the 5' flank of the L1s. These families with L1s exhibiting identity in the 5' flank were eliminated from further analysis, as the L1s may represent the same insertion event that was part of a segmental duplication in the genome whose endpoint happened to coincide with the 3' TSD.
GenBank headers of the appropriate gi record (NT_* contigs) were checked for whether the final, bona fide transduced sequences were included in the annotation of any mRNA or CDS.
Adenine content of L1s
The L1s used to generate the data in Figure 4 represent all L1s that were found in the human genome using RepeatMasker  with the L1.3 sequence as a custom library (see ). To calculate the probability that the length of a stretch of pure A nucleotides in the L1 sequence was less than y, we used the formula:
P(A tract < y) = e^(-nqp y )
where n is the length of the L1 sequence, p is the probability of finding an A in the L1 sequence, and q is (1 - p) .
Additional data files
We are grateful to members of the Boeke lab and Landsman group for helpful discussions during the preparation of this manuscript, especially Liora Strichman-Almashanu, John S.J. Anderson, Wataru Fujibuchi and David Symer. This work was supported by NIH grant CA16519 to J.D.B.
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