Molecular archeology of L1 insertions in the human genome

“The BLASTX program [45] was used to translate our set of full-length L1 DNA sequences in all three reading frames and compare them to the L1.”

“DNA sequence logos [59] of TSDs were generated using binary code downloaded from the site [60].”

“L1 poly(A) tails During the process of TPRT, whereby a copy of an L1 is inserted into the genome, reverse transcription is believed to be primed on the poly(A) tail of the L1 transcript [12,14,17,27].”

“The functional significance of ORF1 is not clear, whereas ORF2 contains three domains critical for L1 propagation: endonuclease [12], reverse transcriptase [7,13], and a 3 ́-terminal Zn finger-like domain [14].”

“The functional significance of ORF1 is not clear, whereas ORF2 contains three domains critical for L1 propagation: endonuclease [12], reverse transcriptase [7,13], and a 3 ́-terminal Zn finger-like domain [14].”

“Furthermore, the concentration of L1s with TSDs on the p arm of the X chromosome is lower than that on the q arm (Figure 8). Lahn and Page [ 52 ] have hypothesized that the distal p arm is the site of the youngest evolutionary strata on the X chromosome, indicating that the X and Y chromosomes actively recombined at t…”

“This modest majority may reflect a weak selection against L1s in the same orientation; such insertions could result in premature truncation of a gene’s mRNA due to the L1 polyadenylation signal [27,48,49].”

“Also, L1-encoded proteins were almost certainly involved in the insertion of most of the Alu elements and processed pseudogenes in the genome [6-10]; thus, L1s are probably responsible, directly or indirectly, for the genesis of most of the transposed fraction of human DNA.”

“The functional significance of ORF1 is not clear, whereas ORF2 contains three domains critical for L1 propagation: endonuclease [12], reverse transcriptase [7,13], and a 3 ́-terminal Zn finger-like domain [14].”

“Nevertheless, this large fraction is a testament to the efficiency with which these elements, now mostly fossil sequences [2-5], have been able to propagate, and it is clear that they played a large part in determining the structure”

“This quality is consistent with previous computational [6] and biochemical [12,34] observations of A-rich target sequences of L1s and Alu sequences.”

“Several instances of disease have been attributed to the disruption of genes by an L1 insertion (for a review see [ 42 ]).”

“It is estimated that 30-60 full-length active L1s reside in the average human genome [ 37 ].”

“A full-length, active human L1 is approximately 6,000 nucleotides long and consists of a 5 ́ UTR with an internal promoter, two open reading frames (ORFs) separated by a 63-nucleotide intergenic region, and a 3 ́ UTR terminating in a poly(A) tail [11] (Figure 1a).”

“It has been suggested that the high concentration of L1s on the X chromosome may have a role in X inactivation of select genes [39].”

“The most variable features of L1s are the poly(A) tail, which has a variable length and can contain simple repeats, the polyadenylation signal, and the 3 ́ UTR, but many changes have also been reported in the coding regions of young L1s, especially in a segment of ORF1 [21-24].”

“(NAD+) gamma, biglycan, tafazzin, deoxyribonuclease I-like 1, and emerin [51].”

“Characteristics of 5 truncated L1 elements The majority of L1s in the human genome are significantly 5 truncated [25]; indeed, only 5% of the 3 intact L1s in our study were full-length (Table 1).”

“L1.3 is one of the most active L1s in a cell-culture assay [37] and is a member of the human-specific Ta-1 subfamily of L1s which encompasses the vast majority of active, young L1 elements in the human genome [ 22 ].”

“Consequently, the 3 TSD of the resulting L1 insertion event can be located hundreds of nucleotides downstream of the L1 sequence itself [29,30].”

“The poly(A) tails found in I factors, a transposable element in Drosophila melanogaster similar to L1s, contain several copies of the simple repeat TAA instead of a traditional poly(A) tail [40,41].”

“Consequently, the 3 TSD of the resulting L1 insertion event can be located hundreds of nucleotides downstream of the L1 sequence itself [29,30].”

“Also, L1-encoded proteins were almost certainly involved in the insertion of most of the Alu elements and processed pseudogenes in the genome [6-10]; thus, L1s are probably responsible, directly or indirectly, for the genesis of most of the transposed fraction of human DNA.”

“Instead, our findings support the recently proposed twin-priming model for inversion [47]; as we showed above, the 3 end topstrand cleavage product has a modest bias to be a ‘T’ nucleotide (Figure 3b) and we indeed see a modest preference for a complementary ‘A’ nucleotide at the corresponding position of the inversio…”

“This quality is consistent with previous computational [6] and biochemical [12,34] observations of A-rich target sequences of L1s and Alu sequences.”

“Also, 3 transduction events associated with L1 insertions add an additional structural variation [27,28]; such elements are generated when the element’s transcript includes some downstream flanking sequences that become transduced, along with the L1 sequence, to a new genomic locus.”

“A new L1 insertion is usually flanked by short direct repeats derived from the target DNA locus upon L1 integration [19]; these repeats are referred to as target site duplications (TSDs), and can range from just a few to more than 200 nucleotides in length [14].”

“This observation is consistent with previous reports that the density of L1s on the X chromosome is almost double that on the autosomes [1,38,39].”

“The progenitor elements of these insertions were always fulllength or near full-length L1s with intact ORFs that were active in retrotransposition assays [11,14,35-37].”

“They propagate by being transcribed from an internal RNA polymerase II promoter [ 15 ,16], and then use their endonuclease and reverse transcriptase respectively to nick a target site and reverse transcribe L1 RNA, integrating the L1 into a new genomic locus [12,17,18]; this process is known as target-site-primed reve…”

“Nevertheless, this large fraction is a testament to the efficiency with which these elements, now mostly fossil sequences [2-5], have been able to propagate, and it is clear that they played a large part in determining the structure”

“An extra 131 nucleotides are found in the 5 UTR of some L1 elements, starting at nucleotide position 777 [26].”

“Nevertheless, this large fraction is a testament to the efficiency with which these elements, now mostly fossil sequences [2-5], have been able to propagate, and it is clear that they played a large part in determining the structure”

“The most variable features of L1s are the poly(A) tail, which has a variable length and can contain simple repeats, the polyadenylation signal, and the 3 ́ UTR, but many changes have also been reported in the coding regions of young L1s, especially in a segment of ORF1 [21-24].”

“The functional significance of ORF1 is not clear, whereas ORF2 contains three domains critical for L1 propagation: endonuclease [12], reverse transcriptase [7,13], and a 3´-terminal Zn finger-like domain [14].”

“The progenitor elements of these insertions were always fulllength or near full-length L1s with intact ORFs that were active in retrotransposition assays [11,14,35-37].”

“3 reference sequence, ensuring the presence of an intact L1 internal promoter [16,43,44].”

“There is evidence that the related sequence-specific R1 element endonuclease makes both cuts in the target DNA [18].”

“Also, L1-encoded proteins were almost certainly involved in the insertion of most of the Alu elements and processed pseudogenes in the genome [6-10]; thus, L1s are probably responsible, directly or indirectly, for the genesis of most of the transposed fraction of human DNA.”

“[6,8,53].”

“3 reference sequence, ensuring the presence of an intact L1 internal promoter [16,43,44].”

“Finally, residues 472(G) and 474(D) in the conserved Z motif in ORF2 are required for reverse transcriptase activity [46].”

“The functional significance of ORF1 is not clear, whereas ORF2 contains three domains critical for L1 propagation: endonuclease [12], reverse transcriptase [7,13], and a 3 ́-terminal Zn finger-like domain [14].”

“This modest majority may reflect a weak selection against L1s in the same orientation; such insertions could result in premature truncation of a gene’s mRNA due to the L1 polyadenylation signal [27,48,49].”

“Indeed, as a result of TPRT and decay over time, most L1 copies are inactivated by truncations, internal rearrangements and mutation”

“Most LINE-1 s in the human genome are grossly truncated at their 5′-end [6, 36, 37].”

“This explains, at least in part, the distribution of predominantly non-functional, 5′ truncated L1s in the genom”

“Although the relatively high incidence of these unusual rearrangements may reflect the cellular milieu of the transformed cells used for these studies, molecular and bioinformatic studies have identified rare deletion events associated with L1 and Alu insertions in humans [35–38].”

“Multiple reports have described the ratio of human L1s that are truncated, we used these reports as the basis for adjusting raw read depth to normalized read depth 53,54 .”

“5A,B; Szak et al. 2002).”

“…the evolutionary history and genomic impact of mammalian retrotransposons has been the subject of intensive investigation (for examples, see Smit et al. 1995; Kapitonov and Jurka 1996; Szak et al. 2002; Xing et al. 2003; Price et al. 2004; Khan et al. 2006; for review, see Deininger et al. 2003).”

“The vast majority of LINE1 elements, however, are truncated or highly degenerate, lack the promoter sequence, and are not capable of active retrotransposition (26, 36 ).”

“For example, TSDs can be used to refine the precise location of TEs, as is implemeted in TSDfinder [75] or SINEDR [76].”

“Only ~3500-7000 are full length, retaining their promoters and hence, the potential ability to lend these promoters to nearby genes [71, 72].”

“A signature-based tool searches a sequence for features comprising the signature of the class of interest [21–24].”

“Known as 39- transduction, this process is thought to result from the read- through of the weak L1 poly(A) signal and is estimated to occur in 15%–23% of all genomic L1s (Holmes et al. 1994; Moran et al. 1999; Goodier et al. 2000; Pickeral et al. 2000; Szak et al. 2002 ).”

“We found variable TSDs of 8–19 bp in length which correspond well to the TSD lengths of preexisting genomic L1 insertions ranging from 9 to 27 bp (15).”

“2 in Szak et al. 2002).”

“Programs for detecting non-LTR retrotransposons The programs TSDFINDER (Szak et al., 2002), SINEDR (Tu et al., 2004) and RTANALYZER (Lucier et al., 2007) have been designed to detect non-LTR retrotransposons.”

“Analyses of endogenous L1s in the human genome [Jurka, 1997; Lander et al., 2001; Szak et al., 2002] further confirmed that L1 integrants carry characteristic features of TPRT integration: L1 sequences generally integrate at 50 TTTT0AA 30 sites, contain an intact 30 end region ending in a polyA,…”

“However, protein-coding sequences are a small target (only 1% of the genome((3))) and thus most new L1 integrations are in introns or intergenic regions.((7)) The consequences of these L1 insertions on gene function have not been thoroughly studied.”

“5 (full-length) AGAAAAATCTTTTCTT (16) TTCT/g 60 4 SVA.”

“The insertion is 3′ intact (contains a polyA-tail) and is flanked by TSDs.”

“TSDFINDER was developed to refine the coordinates of long interspersed nuclear element (LINE) L1 insertions by identifying flanking target site duplication (TSD) sequences and the poly(A) tails of 3′ intact L1 insertions in the human genome [21].”

“Consistent with previous observations (Szak et al., 2002), genic L1 copies are more often found in the antisense orientation (Figure 4—figure supplement 3a).”

“Similar compositions were reported for human L1 polyA tails [35].”

“As to chromosomal distribution, the majority of the L1IMDs were located on chromosomes 1–12, which probably relates to both the larger size of these chromosomes and their higher density of truncated (30 intact) L1 insertions (19).”

“insertions lack these features and may instead contain large deletions, cDNA transduction or 30 element truncation (13,14,24).”

“Some software tools have been developed specifically to detect TSDs [28, 29].”

“LINE elements had much longer TSDs and much broader TSD distributions compared with other retrotransposon super-families (Figure 4a), which is consistent with previous findings about the L1 element in the human genome (35,36).”

“This bias is in good agreement with previous reports that indicated preferential placement of L1 sequences in antisense orientation of host genes with a ratio of 2 (52).”

“Although Figure 1 shows full-length elements, nonLTR retrotransposons in genomes are often 5' truncated due to incomplete reverse transcription [35].”

“…(one exception, 42 bp), a variable-length poly(A) tail of 2–72 nucleotides following putative polyadenylation signals, and a consensus target sequence AA[TTTT. Poly(A) tails were composed of patterned repeats (Szak et al. 2002) in 60 cases, while homogenous poly(A) tails were found in 56 elements.”

“sistent with a previously described preference of L1 for AT-rich regions (Supplemental Table 4; Szak et al. 2002; Boissinot et al. 2004; Gasior et al. 2007).”

“Only 15% of all intact L1 insertions flanked by target site duplications (TSDs) represent full-length insertions, 85% are 5 truncated, and 19% are both 5 -truncated and 5 -inverted (Szak et al. 2002).”

“It has previously been observed that target site duplications and deletions tend to either be 15 nt or 9 nt in length [33], more specifically we observe that the majority of target site duplications are 14 nt in length, and target site deletions have a median of 7 nt (Figure 2B).”

“Since the majority of L1 insertions are 50 truncated (25,26), the length of a reporter gene could potentially be a critical factor in determining the overall L1 activity that can be observed.”

“2005), and for genomic L1s and Alus (Jurka 1997; Myers et al. 2002; Szak et al. 2002).”

“Evolutionary dynamics in a novel L2 clade of non-LTR retrotransposons in Deuterostomia.”

“In summary, we conclude that Alu elements share little in common in terms of functional biases with either older or younger mobile elements, and we can therefore rule out the tolerance and preferential insertion hypotheses, a conclusion that is in fact consistent with previous findings [23,24].”

“Specifically, the loci included in this analysis after experimental confirmation of the computational output possessed all four of the following characteristics: 30 truncation beyond 25 bp (i.e., 5 bp more than the minimum truncation level set during computational screening) relative to the L1HS_30end consensus from t…”

“They frequently give birth to microsatellites upon and after their integration; however, unlike Alus, L1s do so evenly throughout their length, potentially because their whole sequence [and not just their 39-poly(A) tails (Boeke 1997)] is AT-rich (Szak et al. 2002).”

“Other studies have previously reported that T-richness extends beyond four nucleotides upstream of the cleavage site [48,50].”

“Because L1 elements are often truncated or rearranged (Smit et al., 1995; Szak et al., 2002), we based our analyses of L1 subfamily diversity and relationships on 864 bp-long sequences encompassing the last 665 bp of ORF2 and the entire 3′ UTR, to maximize the number of elements included in the analyses.”

“In fact, the majority of genomic L1s are 5) truncated while the 3) ends remain relatively constant (Hutchison et al., 1989; Boissinot et al., 2001; Szak et al., 2002).”

“LINE elements are transposable elements that comprise 21% of the human genome, 16% of the dog genome, and often insert into intronic regions [44,45].”

“With increased interest in TEs themselves, computational tools expanded in their scope, Table 1 Updated bioinformatics tools and databases since those summarized in Lerat (2009) Category Approach used Application name Defining characteristics References TE discovery programs Library-based approaches TransposonPSI Uses…”

“However, reverse transcriptase may not always reach the 5’ end of mRNA [46].”

“In LINEs, target-primed reverse transcription (TPRT) is an inefficient process which often terminates before copying the entire element, leading to fragments truncated at their 5′ end (Szak et al. 2002; Han 2010).”

“In contrast, the E6 protein from β-human papillomavirus (β-HPV 5 and 8) reduces ATM protein levels and attenuates L1 retrotransposition, suggesting that ATM is needed for efficient L1 retrotransposition (Wallace et al., 2013).”

“As a majority of full-length intragenic human L1s are oriented AS to flanking genes’ ORFs (23,24), resulting AS L1 RIFTs frequently include downstream spliced exons expressed in the canonical sense orientation.”

“Only 80–100 human L1 copies are fulllength and retrotransposition-competent [3], whereas the majority of L1 insertions is truncated at their 5′ end and thus not mobile [4].”

“The spacing observed in our assay is within the reported lengths of in vivo LINE-1 TSD events (23, 24).”

“The TSDfinder program [23,37,38] was run on the RepeatMasker *.”

“These rates are much lower than those for TPRT-mediated insertions which encompass the vast majority of Alu insertions in primates [47].”

“A similar insertion site preference was observed for 2 kb sequence blocks as well as for the previously reported L1 Ta subfamily(24) and other L1 subfamilies.(38) In addition, we also analyzed preTa L1 elements inserted in repetitive sequences and Table 1.”

“The signatures of this L1 integration include 5′ truncation, shortening of oligo(dA) at the 3′ end, and target site duplications of 7–20 base pairs in length (48).”

“Each mRNA/EST hit of 99% identity, EST match length of 300 bp, was then compared using BLAST2SEQ to the corresponding HMG mRNA and considered as evidence for RPC expression only if it was 97% identical to the HMG mRNA and therefore was not likely to be its transcript.”

“The lengths of the TSDs flanking the genomic copies of L1PAs (Szak et al. 2002), BovB (Nijman et al. 2002), and RTE_Mdo (Gentles et al. 2007) were analyzed previously; however, these analyses did not include TSDs of 10 bp because the short direct repeats identified could not be statistically…”

“In such a situation RepeatMasker may annotate multiple sequence similarity hits to the given TE family without establishing the common origin of these sequences [21–24], and no information on the temporal order of insertion of overlapping repetitive elements is obtained without human analysis.”

“L1 and Alu are both nonsite-specific and demonstrate a strong bias for insertion into a short, A+T-rich L1 endonuclease target site, as has been demonstrated for genomic L1 and Alu insertions (Boissinot et al., 2000, 2004; Feng et al., 1996; Jurka, 1997; Jurka and Klonowski, 1996; Morrish et al., 2002; Myers et al., 2…”

“Over 500,000 copies of LINE-1 are present in the human genome, of which the vast majority is 5′ truncated [11]; about 3,000copies are full length and 80–100 of these are active re t ro t ransposons [12] .”

“Human L1 and Alu are usually flanked by ~15-bp TSDs [9,33,34].”

“Mammalian LINE-1 elements, which transpose by a similar mechanism, generate variable-sized TSDs, with lengths centering around 9 and 15 bp and never exceeding 60 bp in more than 16,000 analyzed L1 insertions in the human genome (Szak et al., 2002).”

“However, 85% of LINE-1 elements are truncated in their 5′ sequences [20], and thus, PCR-based assays only assess 15% of the LINE-1 elements in the human genome.”

“…species, and even on much smaller scales, could have a catastrophic effect on the evolutionary trajectory of L1s in a genome, especially given the poor success rates of the L1 retrotransposition mechanism in producing new fully functional L1 copies (Kazazian and Moran 1998; Szak et al. 2002).”

“Thus, TSD enables us to determine the target-site boundaries of genomic L1 copies (Szak et al. 2002; Martin et al. 2005b; Zingler et al. 2005).”

“This experimental evidence has been corroborated by computer analyses of the sites of preexisting L1 and Alu element insertions in the human genome (Jurka, 1997; Szak et al., 2002).”

“In various contexts, transposable elements have been shown to affect the expression of genes in their vicinity [58,59], and some elements are included in the untranslated regions of host mRNAs [60,61].”

“However, LINE retrotransposition is often truncated at the 5’ end [17].”

“In the case of LINE-1, however, only a minor fraction (about 80–100) of all copies present in the human genome are full-length and retrotranspositionally competent [4], whereas the vast majority of genomic LINE-1 copies are truncated at their 5’ end and thus non-mobile [5], yet still transcriptionally competent: this…”