Heads or tails: L1 insertion-associated 5’
Thomas J Meyer†, Deepa Srikanta†, Erin M Conlin, Mark A Batzer*
Background: L1s are one of the most successful autonomous mobile elements in primate genomes. These
elements comprise as much as 17% of primate genomes with the majority of insertions occurring via target
primed reverse transcription (TPRT). Twin priming, a variant of TPRT, can result in unusual DNA sequence
architecture. These insertions appear to be inverted, truncated L1s flanked by target site duplications.
Results: We report on loci with sequence architecture consistent with variants of the twin priming mechanism and
introduce dual priming, a mechanism that could generate similar sequence characteristics. These insertions take
the form of truncated L1s with hallmarks of classical TPRT insertions but having a poly(T) simple repeat at the 5’
end of the insertion. We identified loci using computational analyses of the human, chimpanzee, orangutan, rhesus
macaque and marmoset genomes. Insertion site characteristics for all putative loci were experimentally verified.
Conclusions: The 39 loci that passed our computational and experimental screens probably represent inversion-
deletion events which resulted in a 5’ inverted poly(A) tail. Based on our observations of these loci and their local
sequence properties, we conclude that they most probably represent twin priming events with unusually short
non-inverted portions. We postulate that dual priming could, theoretically, produce the same patterns. The
resulting homopolymeric stretches associated with these insertion events may promote genomic instability and
create potential target sites for future retrotransposition events.
Retrotransposons, mobile elements that move via a ‘copy
and paste’ mechanism, called retrotransposition, are ubi-
quitous in primate genomes [1,2]. L1s, members of the
long interspersed element (LINE) family of non-long
terminal repeat (LTR) retrotransposons, which comprise
as much as ~17% of primate genomes, are present in
copy numbers of approximately 520,000 and have
actively molded primate genomic architecture for the
last 65 million years [3-5]. During their mobilization,
they generate insertions containing L1 sequence and, in
some cases, transduced sequence and deletion of adja-
cent genomic sequence [6-9]. Long after insertion, how-
ever, L1s can serve as sites of non-allelic homologous
recombination, resulting in the loss, gain and inversion
of genetic material [10,11]. In these ways, L1s have been
shown to disrupt genes, cause disease states and contri-
bute to the expansion and contraction of the genome
These autonomous retrotransposons contain a 5’
untranslated region (UTR) with an RNA polymerase II
promoter, two open reading frames (ORFs), and a 3’
UTR encompassing a poly(A) tail; full-length L1s are ~6
kb long . ORF1 encodes an RNA-binding protein
with nucleic acid chaperone activity and ORF2 encodes
both a reverse transcriptase (RT) and an endonuclease
(EN) [16-19]. The L1 EN and RT are integral to an
insertion process, termed target primed reverse tran-
scription (TPRT), used by L1s to insert de novo copies
of themselves into their host genomes  (Figure 1a).
Non-autonomous retrotransposons, like Alu and SVA
elements, use the L1 retrotransposon enzymatic machin-
ery for their own mobilization via TPRT [21,22].
The classical TPRT mechanism involves a single nick
on the bottom strand at a loosely-preferred cleavage
motif (foe example, 5’-TTTT/A-3’) by the EN, leaving a
free 3’ hydroxyl group at the nick site. The L1 mRNA
* Correspondence: firstname.lastname@example.org
† Contributed equally
Department of Biological Sciences, Biological Computation and Visualization
Center, Louisiana State University, 202 Life Sciences Bldg, Baton Rouge, LA
Meyer et al. Mobile DNA 2010, 1:7
© 2010 Meyer et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
then anneals to the nick using its poly(A) tail and L1 RT
uses this mRNA as a template for reverse transcription
beginning at the free 3’ hydroxyl group. Top strand clea-
vage, integration of the cDNA, and synthesis of a top
strand complement to the cDNA complete the insertion,
leaving the structural hallmarks of classical TPRT: intact
target site duplications (TSDs), a typical EN cleavage
site motif, anda variable
[17,20,23,24]. While full-length L1s are ~6 kb in length,
many L1 insertions are 5’ truncated (averaging ~900 bp
in length) and no longer able to actively retrotranspose
[13,15,24,25]. Anomalies observed in TPRT-inserted
copies have led to the proposal of variant mechanisms,
such as internal and twin priming, that account for non-
standard sequence architecture for TPRT-inserted ele-
ments (Figure 1b) [9,26-29]. Recent studies have shown
that insertions using twin priming lead to new retrogene
formation, limit L1 expansion and cause genome
A recent human genome-wide analysis led to the dis-
covery of homopolymeric thymine (poly(T)) stretches
just upstream of truncated L1 insertions . Intrigued
by these homopolymeric stretches associated with loci
having many hallmarks of classical TPRT, we performed
Figure 1 Classical target primed reverse transcription (TPRT), twin priming, variants of twin priming and dual priming mechanisms. (a)
A schematic of classical TPRT. The poly(A) tail of an L1 mRNA anneals to the target site created by L1 endonuclease. L1 reverse transcription (RT)
primes at the target site and synthesizes the bottom-strand cDNA. A subsequent second-strand nick and synthesis results in an L1 insertion with
a 3’ poly(A) flanked by TSDs. (b) Twin priming. In this variant of TPRT, after the second-strand nick, a site internal to the mRNA anneals to the
top strand overhang. A second RT molecule primes at this site, generating an inverted L1 cDNA. (c) This twin priming variant involves the
disengagement of the first RT before reaching the end of the poly(A) tail, resulting in an insertion with a 5’ poly(T) stretch, but lacking a 3’ poly
(A) tail. Like classical twin priming, this mechanism results in an inverted L1 structure. (d) A second twin priming variant creates an insertion with
both a 3’ poly(A) tail and a 5’ poly(T) stretch. The first RT falls off before reaching the end of the poly(A) tail. (e) Dual priming. Classical TPRT
involving the first mRNA begins on the first strand. After the second strand nick, a second mRNA anneals to the second strand and undergoes
classical TPRT. Note that this panel is rotated 180° relative to the orientation of all other panels. This is done to show that the resulting insertion
will appear the same to computational filters as the above twin priming variant.
Meyer et al. Mobile DNA 2010, 1:7
Page 2 of 12
directly from PCR products and were cloned into vec-
tors using the TOPO TA (fragments <2 kb) cloning kit
(Invitrogen). Following cloning, two to four colonies
were randomly selected for colony PCR. Those colonies
that appeared to contain the insert were then mini-
prepped using the manufacturer’s protocol (5PRIME).
Sequencing results were obtained using an ABI3130XL
automated DNA sequencer and analysed using BioEdit
http://www.mbio.ncsu.edu/BioEdit/page2.html and the
SeqMan and EditSeq utilities from the DNAStar® V.5
software package. Close inspection of the flanking
sequence and the results of PCR were used to confirm
the pre-insertion sequence for each locus from a mini-
mum of one outgroup genome. Sequences generated in
this study have been deposited in GenBank under
Accession Nos GQ477185-GQ477273.
Microhomology and L1 endonuclease cleavage site
The 6 bp of the 3’ TSD closest to the insert were com-
pared to the corresponding sequence at those positions
in an alignment of each candidate L1 fragment to the
L1 consensus in the manner described in Sen et al. .
The 3’ junctions of some loci were excluded from analy-
sis if a non-candidate L1 sequence was included in the
insert. At the internal junction between the poly(T)
stretch and the 5’ end of the candidate L1, the first 6 bp
of the L1 were compared to the last 6 bp of the poly(T)
and the internal junction of a locus was excluded if any
non-candidate L1 sequence was found between the poly
(T) stretch and candidate L1.
EN cleavage site analysis of the 3’ target site of each
locus for similarity to the preferred L1 EN cleavage
motif (5’-TTTT/A-3’) was carried out by comparing this
motif to the first four bases of the reverse complemen-
ted TSD and the first base of the flanking sequence. Dif-
ferences in base composition were scored with
transitions given a weight of 0.5 and transversions given
a weight of 1.0 [8,33]. The frequency of divergence from
the L1 EN cleavage site was then calculated.
The above analyses were performed on the loci with
the candidate L1s in the sense orientation. In order to
investigate the possibility that the candidate L1s were
inserted in the antisense orientation, both microhomol-
ogy and EN cleavage site analyses were repeated on the
reverse complements of our sequences. In these cases,
the 5’ junctions closest to the poly(T) stretches were
analysed as if they were 3’ poly(A) stretches.
EN: endonuclease; LINE: long interspersed element; LTR: long terminal
repeat; NHEJ: non-homologous end joining; ORF: open reading frame; PCR:
polymerase chain reaction; RT: reverse transcriptase; TPRT: target primed
reverse transcription; TSD: target site duplication; UTR: untranslated region.
The authors would like to thank all members of the Batzer laboratory for
their advice and feedback. They would especially like to thank J A Walker, K
Han, M K Konkel and K Engel, for their suggestions and advice, and C Faulk
and D Donze for their useful comments during the preparation of the
manuscript. The authors are grateful to LSU BioGrads for their assistance (No.
09-15; TJM). The authors also thank the Genome Center at Washington
University School of Medicine in St Louis for producing the common
marmoset genome data used in this study, which can be obtained from
Callithrix_jacchus-2.0.2/. This research was supported by National Institutes of
Health RO1 GM59290 (MAB) and the State of Louisiana Board of Regents
Support Fund (MAB).
TJM, DS and MAB designed the research; TJM, DS and EMC performed the
research; MAB contributed new reagents/analytic tools; TJM, DS and EMC
analysed the data; and TJM, DS and MAB wrote the paper.
The authors declare that they have no competing interests.
Received: 18 August 2009
Accepted: 1 February 2010 Published: 1 February 2010
1. Smit AF, Toth G, Riggs AD, Jurka J: Ancestral, mammalian-wide
subfamilies of LINE-1 repetitive sequences. J Mol Biol 1995, 246:401-417.
2.Cordaux R, Batzer MA: The impact of retrotransponsons on human
genome evolution. Nature Reviews Genetics 2009, 10:691-703.
3. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K,
Dewar K, Doyle M, FitzHugh W, et al: Initial sequencing and analysis of
the human genome. Nature 2001, 409:860-921.
4. Brouha B, Schustak J, Badge RM, Lutz-Prigge S, Farley AH, Moran JV,
Kazazian HH Jr: Hot L1s account for the bulk of retrotransposition in the
human population. Proc Natl Acad Sci USA 2003, 100:5280-5285.
5. Smit AF: The origin of interspersed repeats in the human genome. Curr
Opin Genet Dev 1996, 6:743-748.
6. Moran JV, Holmes SE, Naas TP, DeBerardinis RJ, Boeke JD, Kazazian HH Jr:
High frequency retrotransposition in cultured mammalian cells. Cell 1996,
7.Moran JV, DeBerardinis RJ, Kazazian HH Jr: Exon shuffling by L1
retrotransposition. Science 1999, 283:1530-1534.
8. Han K, Sen SK, Wang J, Callinan PA, Lee J, Cordaux R, Liang P, Batzer MA:
Genomic rearrangements by LINE-1 insertion-mediated deletion in the
human and chimpanzee lineages. Nucleic Acids Res 2005, 33:4040-4052.
9. Gilbert N, Lutz-Prigge S, Moran JV: Genomic deletions created upon LINE-
1 retrotransposition. Cell 2002, 110:315-325.
10. Han K, Lee J, Meyer TJ, Remedios P, Goodwin L, Batzer MA: L1
recombination-associated deletions generate human genomic variation.
Proc Natl Acad Sci USA 2008, 105:19366-19371.
11. Lee J, Han K, Meyer TJ, Kim HS, Batzer MA: Chromosomal inversions
between human and chimpanzee lineages caused by retrotransposons.
PLoS One 2008, 3:e4047.
12.Belancio VP, Hedges DJ, Deininger P: LINE-1 RNA splicing and influences
on mammalian gene expression. Nucleic Acids Res 2006, 34:1512-1521.
13.Konkel MK, Wang J, Liang P, Batzer MA: Identification and characterization
of novel polymorphic LINE-1 insertions through comparison of two
human genome sequence assemblies. Gene 2007, 390:28-38.
14. Oliver KR, Greene WK: Transposable elements: powerful facilitators of
evolution. Bioessays 2009, 31:703-714.
15. Kazazian HH Jr, Moran JV: The impact of L1 retrotransposons on the
human genome. Nat Genet 1998, 19:19-24.
16. Mathias SL, Scott AF, Kazazian HH Jr, Boeke JD, Gabriel A: Reverse
transcriptase encoded by a human transposable element. Science 1991,
17. Feng Q, Moran JV, Kazazian HH Jr, Boeke JD: Human L1 retrotransposon
encodes a conserved endonuclease required for retrotransposition. Cell
Meyer et al. Mobile DNA 2010, 1:7
Page 11 of 12
18. Kolosha VO, Martin SL: In vitro properties of the first ORF protein from
mouse LINE-1 support its role in ribonucleoprotein particle formation
during retrotransposition. Proc Natl Acad Sci USA 1997, 94:10155-10160.
Jurka J: Sequence patterns indicate an enzymatic involvement in
integration of mammalian retroposons. Proc Natl Acad Sci USA 1997,
Luan DD, Korman MH, Jakubczak JL, Eickbush TH: Reverse transcription of
R2Bm RNA is primed by a nick at the chromosomal target site: a
mechanism for non-LTR retrotransposition. Cell 1993, 72:595-605.
Batzer MA, Deininger PL: Alu repeats and human genomic diversity. Nat
Rev Genet 2002, 3:370-379.
Ostertag EM, Goodier JL, Zhang Y, Kazazian HH Jr: SVA elements are
nonautonomous retrotransposons that cause disease in humans. Am J
Hum Genet 2003, 73:1444-1451.
Fanning TG, Singer MF: LINE-1: a mammalian transposable element.
Biochim Biophys Acta 1987, 910:203-212.
Szak ST, Pickeral OK, Makalowski W, Boguski MS, Landsman D, Boeke JD:
Molecular archeology of L1 insertions in the human genome. Genome
Biol 2002, 3:research0052.
Myers JS, Vincent BJ, Udall H, Watkins WS, Morrish TA, Kilroy GE,
Swergold GD, Henke J, Henke L, Moran JV, Jorde LB, Batzer MA: A
comprehensive analysis of recently integrated human Ta L1 elements.
Am J Hum Genet 2002, 71:312-326.
Ostertag EM, Kazazian HH Jr: Twin priming: a proposed mechanism for
the creation of inversions in l1 retrotransposition. Genome Res 2001,
Kazazian HH Jr, Goodier JL: LINE drive. retrotransposition and genome
instability. Cell 2002, 110:277-280.
Kulpa DA, Moran JV: Cis-preferential LINE-1 reverse transcriptase activity
in ribonucleoprotein particles. Nat Struct Mol Biol 2006, 13:655-660.
Srikanta D, Sen SK, Conlin EM, Batzer MA: Internal priming: An
opportunistic pathway for L1 and Alu retrotransposition in hominins.
Gene 2009, 448(2):233-41.
Kojima KK, Okada N: mRNA retrotransposition coupled with 5’ inversion
as a possible source of new genes. Mol Biol Evol 2009, 26:1405-1420.
Roth DB, Chang XB, Wilson JH: Comparison of filler DNA at immune,
nonimmune, and oncogenic rearrangements suggests multiple
mechanisms of formation. Mol Cell Biol 1989, 9:3049-3057.
Goodman M, Porter CA, Czelusniak J, Page SL, Schneider H, Shoshani J,
Gunnell G, Groves CP: Toward a phylogenetic classification of Primates
based on DNA evidence complemented by fossil evidence. Mol
Phylogenet Evol 1998, 9:585-598.
Zingler N, Willhoeft U, Brose HP, Schoder V, Jahns T, Hanschmann KM,
Morrish TA, Lower J, Schumann GG: Analysis of 5’ junctions of human
LINE-1 and Alu retrotransposons suggests an alternative model for 5’-
end attachment requiring microhomology-mediated end-joining.
Genome Res 2005, 15:780-789.
Sen SK, Huang CT, Han K, Batzer MA: Endonuclease-independent insertion
provides an alternative pathway for L1 retrotransposition in the human
genome. Nucleic Acids Res 2007, 35:3741-3751.
Crooks GE, Hon G, Chandonia JM, Brenner SE: WebLogo: a sequence logo
generator. Genome Res 2004, 14:1188-1190.
Levinson G, Gutman GA: Slipped-strand mispairing: a major mechanism
for DNA sequence evolution. Mol Biol Evol 1987, 4:203-221.
Schlotterer C, Tautz D: Slippage synthesis of simple sequence DNA.
Nucleic Acids Res 1992, 20:211-215.
Arcot SS, Wang Z, Weber JL, Deininger PL, Batzer MA: Alu repeats: a
source for the genesis of primate microsatellites. Genomics 1995,
Kiss AM, Jady BE, Bertrand E, Kiss T: Human box H/ACA pseudouridylation
guide RNA machinery. Mol Cell Biol 2004, 24:5797-5807.
Garcia PB, Robledo NL, Islas AL: Analysis of non-template-directed
nucleotide addition and template switching by DNA polymerase.
Biochemistry 2004, 43:16515-16524.
Gilbert N, Lutz S, Morrish TA, Moran JV: Multiple fates of L1
retrotransposition intermediates in cultured human cells. Mol Cell Biol
Ricchetti M, Buc H: A reiterative mode of DNA synthesis adopted by HIV-
1 reverse transcriptase after a misincorporation. Biochemistry 1996,
43. Ling J, Zhang L, Jin H, Pi W, Kosteas T, Anagnou NP, Goodman M, Tuan D:
Dynamic retrotransposition of ERV-9 LTR and L1 in the beta-globin gene
locus during primate evolution. Mol Phylogenet Evol 2004, 30:867-871.
Symer DE, Connelly C, Szak ST, Caputo EM, Cost GJ, Parmigiani G, Boeke JD:
Human l1 retrotransposition is associated with genetic instability in vivo.
Cell 2002, 110:327-338.
Suzuki J, Yamaguchi K, Kajikawa M, Ichiyanagi K, Adachi N, Koyama H,
Takeda S, Okada N: Genetic evidence that the non-homologous end-
joining repair pathway is involved in LINE retrotransposition. PLoS Genet
Gottlich B, Reichenberger S, Feldmann E, Pfeiffer P: Rejoining of DNA
double-strand breaks in vitro by single-strand annealing. Eur J Biochem
Deininger PL, Moran JV, Batzer MA, Kazazian HH Jr: Mobile elements and
mammalian genome evolution. Curr Opin Genet Dev 2003, 13:651-658.
Shibata D, Peinado MA, Ionov Y, Malkhosyan S, Perucho M: Genomic
instability in repeated sequences is an early somatic event in colorectal
tumorigenesis that persists after transformation. Nat Genet 1994,
Denver DR, Feinberg S, Estes S, Thomas WK, Lynch M: Mutation rates,
spectra and hotspots in mismatch repair-deficient Caenorhabditis
elegans. Genetics 2005, 170:107-113.
Paoloni-Giacobino A, Chaillet JR: Evolutionary appearance of
mononucleotide repeats in the coding sequences of four genes in
primates. J Genet 2007, 86:279-283.
Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM,
Haussler D: The human genome browser at UCSC. Genome Research 2002,
Smit A, Hubley R, Green P: RepeatMasker Open-3.0. 1996.
Kent WJ: BLAT–the BLAST-like alignment tool. Genome Res 2002,
Rozen S, Skaletsky H: Primer3 on the WWW for general users and for
biologist programmers. Methods Mol Biol 2000, 132:365-386.
Cite this article as: Meyer et al.: Heads or tails: L1 insertion-associated 5’
homopolymeric sequences. Mobile DNA 2010 1:7.
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