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 LINE
GG Schumann, Federal Institute for Vaccines and Biomedicines, Langen, Germany
2013 Elsevier Inc. All rights reserved.
This article is a revision of the previous edition article by L Silver, volume 2, pp 11031104, 2001, Elsevier Inc.
Glossary
Endonuclease Enzyme that cleaves the phosphodiester
bond within a polynucleotide chain.
Processed pseudogenes Result from reverse transcription
of cellular messenger RNAs (mRNAs) mediated by the
long interspersed element (LINE)-encoded protein
machinery. They are devoid of intron sequences.
Retrotransposition A copy-and-paste mechanism
whereby a retrotransposable element is copied from one
Introduction
Long interspersed elements (LINEs) are mobile genetic ele
ments in mammalian genomes that are members of the
group of retrotransposons which all replicate via an RNA inter
mediate by a copy-and-paste mechanism. Retrotransposons
can be divided into two classes: retrotransposons that are
flanked by long terminal repeats (LTRs) and retrotransposons
without LTRs, named non-LTR retrotransposons. LINEs, short
interspersed nuclear elements (SINEs), and processed pseudo-
genes represent families of non-LTR retrotransposons. LINEs
are termed autonomous because full-length LINEs encode the
protein machinery which they use for their own replication.
Nonautonomous non-LTR retrotransposons, like SINEs, have
evolved refined parasitic strategies to recruit LINE-encoded
proteins for their own mobilization. Group II introns that are
present in mitochondria and chloroplasts of plants and fungi
are assumed to be the ancestors of autonomous non-LTR
retrotransposons.
LINEs are replicating repetitive elements that are 67 kb in
length, possess an internal RNA polymerase II promoter, and
include two open reading frames (ORFs) that are essential for
the replication process and a polyA tail. The first ORF (ORF1)
codes for a protein that binds nucleic acids, while ORF2 encodes
an endonuclease (EN) and a reverse transcriptase (RT) domain,
and a function associated with a cysteinehistidine-rich motif
at the C-terminal end of the ORF2 protein (Figure 1).
LINE-encoded EN domains belong to a wide family of structu
rally related phosphohydrolases most closely related to apurinic/
apyrimidinic (AP) DNA repair ENs. Therefore, LINEs are mem
bers of the group termed apurinic/apyrimidinic EN (APE)-type
non-LTR retrotransposons (APE-type elements) that comprise
most identified retrotransposons. EN domains encoded by
human, mouse, and rat L1 elements recognize and cleave the
loose genomic target sequence 5-TTTT/A-3 and are therefore
designated semi-specific. Only a small subset of APE-type ele
ments insert in a site-specific manner, that is, either sequence
specifically into defined target sequences or position specifically
into loci with a defined distance to certain repetitive multicopy
genes. On the basis of both the phylogenetic relationship of their
RT sequences and the nature and arrangement of their protein
246 Brenners Encyclopedia of Genetics, 2nd edition, Volume 4
genomic location and inserted into another genomic
location, using a ribonucleic acid intermediate.
Retrotransposon Transposable DNA element that
replicates in the host genome through reverse
transcription of the encoded RNA into complementary
DNA (cDNA) that is inserted at a new genomic site.
Reverse transcriptase (RNA-dependent DNA
polymerase) A DNA polymerase enzyme that transcribes
single-stranded RNA into single-stranded cDNA.
domains, APE-type elements can be divided into 4 groups and
11 clades. Clades L1 and L2 include mammalian LINEs termed
L1 and L2. There are full-length and 5-truncated LINEs, and they
are flanked by short direct repeats of varying lengths (920 bp)
which are termed target site duplications (TSDs). L1 elements are
the most successful and best-characterized LINEs to date, and
represent the most common and only active LINE family in the
human genome.
The Human LINE-1 Retrotransposon
LINE-1 retotransposons have proliferated over the past 80 mil
lion years of primate evolution and now comprise more than
500 000 L1 copies per haploid human genome. More than
one-third of the genome is the result, directly or indirectly, of
L1 retrotransposon activity because the L1-encoded protein
machinery also mediated retrotransposition of the nonautono
mous Alu and SVA elements and processed pseudogene
formation. The average human diploid genome has 80100
active L1s that are able to autonomously replicate by retro
transposition (Figure 2). At least 1 in every 212 human
newborns has a new genomic L1 insertion that occurred in
parental germ cells or in early embryonic development. L1
insertions account for 0.3% of all mutations in the human
genome. Although L1s have a marked cis preference, whereby
their proteins greatly prefer to act on the RNA that encodes
them, they are still able on occasion to mobilize nonautono
mous sequences in trans. trans-Mobilization of Alu elements
resulted in the expansion of Alu sequences to 1.1 million copies
in the human genome. Alu retrotransposition events occur in at
least 1 in every 21 individuals. Alu insertions have accounted
for over 43 cases of human genetic or tumorigenic disease.
L1-mediated retrotransposition can occur in germ cells,
embryonic stem cells, neural progenitor cells, and tumor
cells and occasionally in differentiated somatic cell types. L1
elements continuously alter genome structure in many ways,
both destructive and constructive, and their activity can cause
genomic instability that can lead to human disease. Beyond
their influence on genome evolution on the species level,
LINE-mediated retrotransposition reshuffles individual
doi:10.1016/B978-0-12-374984-0.00869-X
 LINE 247

L1Hs (Homo sapiens)

IGR
5 UTR ORF1 ORF2 3 UTR
TSD APE RT C pA TSD

L1Md (Mus domesticus)

LPR

IGR
5 UTR ORF1 ORF2 3 UTR
TSD APE RT C pA TSD

Figure 1 Schematic organization of functional mammalian LINE-1 retrotransposons. Human and mouse L1 elements are 6 and 7 kb in length,
respectively, and represent the best-characterized mammalian L1 elements. In contrast to L1Hs, the 5 UTR of L1s in rodents is bipartite: there is a 5
module that can occur as a tandem repeat and a 3 module named tether that links the tandem repeat of monomers to ORF1. Also, the 5 one-third of
murine ORF1s contain a hypervariable domain, termed length polymorphic region (LPR). APE, apurinic/apyrimidinic endonuclease domain; C, cysteine/
histidine-rich motif; IGR, intergenic region; ORF, open reading frame; pA, polyA tail; TSD, target site duplication; UTR, untranslated region.

5 UTR ORF1 ORF2 3 UTR poly(A)

RNA-binding protein EN and RT

Export of the RNA

LINE-1 RNA

Transcription Translation

L1ORF1p
Ribosome
Nucleus

Reverse transcription
at the site of integration L1ORF2p

Import of the RNAprotein complex

Cytoplasm

Figure 2 Schematic of the proposed replication cycle of mammalian LINE-1 retrotransposons. A functional genomic full-length L1 element is transcribed.
The resulting L1 transcript (red) is exported to the cytoplasm. ORF1- (green circles) and ORF2-encoded proteins (blue ovals) are translated and bind the L1
RNA molecule that encoded them (cis preference). The L1 RNA and associated proteins return to the nucleus as ribonucleoprotein complex by active transport
or entry during nuclear membrane breakdown at mitosis. The L1 RNA is reverse transcribed and integrated into the genome by a mechanism termed TPRT.
The process depicted results in a DNA copy of the original L1 element at a new genomic location (red bar in chromosome). The new L1 copy often differs from
the original by being truncated or rearranged during the retrotransposition process. Black X-like structures represent chromosomes.

genomes in many different ways, a process that is often asso-
ciated with the induction of local genomic instabilities. LINEs
affect the genome by (1) insertion into genic functional
sequences (promoters, enhancers, exons, etc.) which can
lead to insertional mutagenesis, by causing (2) substantial
deletions, (3) intrachromosomal homologous recombination
between LINEs, (4) transduction of 5- and 3-flanking
sequences during retrotransposition, and by (5) aiding trans-
generation of processed pseudogenes and trans-mobilization
of SINEs. LINE insertions influence genomic gene expression
through (1) transcript pausing or termination at the L1 inser
tion site, (2) alternative splicing, (3) effects of L1 sense/
antisense promoter, and (4) heterochromatization.
So far, 96 cases of human genetic disorders (e.g., hemophilia,
cystic fibrosis, neurofibromatosis) and tumorigenic diseases
(e.g., breast cancer, colon cancer) have been shown to be the
consequence of local genomic instabilities caused by the activity
of the human L1 retrotransposon.
248 LINE
LINE-1 Replication and the Mechanism of
Target-Primed Reverse Transcription
The 5 untranslated region (UTR) of a human full-length L1
element contains an internal promoter that directs transcrip
tion from the 5 end of the element. The internal promoter
structure makes sense for a retrotransposon, which must take
its promoter with it to generate an active copy when it inserts
into a new location. Within the 5 UTR, several transcription
factor-binding sites have been identified. SRY family-binding
sites and an RUNX3-binding site appear to be important for
transcriptional activation, while a YY-1-binding site near
the 5 end directs accurate transcription initiation. After L1
RNA transcription and transport to the cytoplasm, both
L1-encoded proteins ORF1p and ORF2p are translated
(Figure 2). ORF1p is a non-sequence-specific RNA-binding
protein with in vitro nucleic acid chaperone activity. The
ORF2-encoded EN and RT activities have well-defined roles
in the retrotransposition process. L1 proteins and L1 RNA
assemble into a ribonucleoprotein particle that is predomi
nantly cytoplasmic, but presumably only a small amount of it
is transported into the nucleus. Once in the nucleus, L1 pro
teins copy L1 RNA into DNA at the genomic integration site
via a process named target-primed reverse transcription
(TPRT).
During TPRT, ORF2p nicks the first strand of the target DNA
to generate a free 3-hydroxyl group (Figure 3). This hydroxyl
acts as a primer for reverse transcription using L1 RNA as
template. The result is simultaneous reverse transcription and
joining of the 5 end of the first-strand complementary DNA
(cDNA) with the genome after the EN has cleaved the second
Target site
Genomic 5
target DNA
1) First-strand
5
2) Annealing
of RNA
LINE-1 RNA
3
(d) Removal
of RNA
LINE-1 cDNA
3
Microhomology
TSD
Figure 3 Schematic of the TPRT mechanism executed by the LINE-encoded protein machinery (see text). (a) First-strand cleavage; (b) annealing of
RNA; (c) reverse transcription; (d) removal of RNA; (e) second-strand cleavage; (f) initiation of second-strand synthesis; (g) remaining DNA synthesis.
strand of the genomic target site to produce a staggered break.
There is evidence that factors of the host cell-encoded
double-strand break repair pathway are involved in the attach
ment of the 3 end of the first-strand cDNA to the genomic
target DNA via pairing of complementary ends and initiation
of second-strand synthesis. Removal of RNA and completion
of DNA synthesis produce a complete insertion flanked by
TSDs.
Potential Beneficial Effects of LINE-1 Activity
There are several effects of L1 activity that could also be con
structive for the host organism. First, L1s occasionally repair
double-strand breaks in DNA by inserting into the genome via
an EN-independent pathway. Second, retrotransposons pro
vide their sequences for a number of protein-coding exons of
genes. In the human genome, L1 or Alu sequences are present
in nearly 200 confirmed and 2400 predicted protein-coding
sequences. Third, L1 retrotransposition can produce new chi
meric retrogenes that are probably generated through
template switching of L1 RT from L1 RNA or Alu RNA to
other small nuclear RNAs. Fourth, L1 elements can affect
gene expression that can be beneficial for the host organism.
They contain an antisense promoter in their 5 UTR region,
and a number of expressed genes located 5 to full-length L1s
have alternate transcription start sites in this L1 region. Also,
intronic L1 insertions in either orientation can affect the RNA
production of endogenous genes, both qualitatively and
quantitatively.
5
cleavage
Second-strand
cleavage site
3) Reverse
transcription
5) Second-strand
cleavage
6) Pairing of
complementary ends
7) Remaining
DNA synthesis
TSD


Host-Encoded Mechanisms of Defense against LINE-1
Retrotransposition
Given that non-LTR retrotransposons affect the genome in so
many ways, it is not surprising that the host cell has developed
various mechanisms to regulate their activity. Truncation, muta
tion, and rearrangement inactivate L1 insertions. Foremost is
transcript malformation due to splicing and premature polyade
nylation, and methylation of the L1 5 UTR that inhibits L1
transcription. Pausing and premature termination of transcription
inhibits L1 expression. Heterochromatization of L1s suppresses
expression. Double-strand RNA produced by transcription from
both the sense and antisense promoters may inhibit L1 retro
transposition by an RNA interference mechanism. ORF1 protein
and L1 RNA may be sequestered in stress granules in the
cytoplasm. Various APOBEC3 proteins that have cytidine deami
nase activity reduce L1 retrotransposition in cell culture by an
unknown mechanism. The nuclease TREX1 prevents accumula
tion of single-strand DNA that was generated by retrotransposon
mediated reverse transcription.
LINE-1-Mediated Genome Manipulation in Vertebrates
Transposable elements like LINEs are DNA segments with the
unique ability to move about in the genome. This inherent
feature can be exploited to harness these elements as tools for
genome-wide, forward insertional mutagenesis that provides
a powerful and high-throughput means to ascribe functions to
genes associated with particular biological pathways. Insertional
mutagenesis using engineered transposable elements can be one
of the most productive and versatile approaches to disrupt and
manipulate genes on a genome-wide scale. However, even if a
transposable element inserts into a gene, it may not have a
mutagenic effect. For example, intronic insertions are likely
spliced out without having an effect on gene expression. Thus,
various technologies have been established to enhance muta
genicity as well as reporting capabilities of insertional vectors by
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LINE 249
trapping transcription units. Murine LINE retrotransposons
were engineered to be used as tools for recessive loss-of-function
screens in the germ line in vivo. A recently established system for
mutagenesis screens is based on a codon-optimized mouse L1
element termed ORFeus that retrotransposes 200-fold more
frequently than the wild-type mouse L1. This hyperactive syn
thetic L1 element represents a significant technological advance,
because it allows for sufficiently efficient L1-mediated inser
tional mutagenesis. One promising system is based on the
ORFeus transgene driven by a constitutive promoter and marked
by a retrotransposition indicator cassette. Genomic distribution
of ORFeus de novo retrotransposon insertions revealed 28% of
the events occurring in genes.
See also: Processed Pseudogene; Retrotransposons; Reverse
Transcription; SINE; Transposable Elements.
Further Reading
Babushok DV and Kazazian HH, Jr. (2007) Progress in understanding the biology of the
human mutagen LINE-1. Human Mutation 28: 527539.
Beck CR, Garcia-Perez JL, Badge RM, and Moran JV (2011) LINE-1 elements in
structural variation and disease. Annual Review of Genomics and Human Genetics
12: 187215.
Cordaux R and Batzer MA (2009) The impact of retrotransposons on human genome
evolution. Nature Reviews. Genetics 10: 691703.
Craig N, Craigie R, Gellert M, and Lambowitz AM (2002) Mobile DNA II. Washington,
DC: ASM Press.
Eickbush TH and Jamburuthugoda VK (2008) The diversity of retrotransposons and the
properties of their reverse transcriptases. Virus Research 134: 221234.
Goodier J and Kazazian HH, Jr. (2008) Retrotransposons revisited: The restraint and
rehabilitation of parasites. Cell 135: 2335.
Hancks DC and Kazazian HH, Jr. (2012) Active human retrotransposons: variation and
disease. Current Opinion in Genetics & Development 22: 113.
Ivics Z, Ma L, Mts L, et al. (2009) Transposon-mediated genome manipulation in
vertebrates. Nature Methods 6: 415422.
Kazazian HH, Jr. (2004) Mobile elements: Drivers of genome evolution. Science 303:
16261632.
Volff J-N (2005) Retrotransposable Elements and Genome Evolution. Basel: Karger.