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J Biomed Biotechnol. 2006; 2006: 32713. Published online 2006 March 20. doi: 10.1155/JBB/2006/32713. | PMCID: PMC1559915 |
The Potential Regulation of L1 Mobility by RNA Interference Shane R. Horman,1 Petr Svoboda,2 and Eline T. Luning Prak1* 1Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6055, USA 2Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland Received August 6, 2005; Revised December 12, 2005; Accepted December 20, 2005. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited. The hypothesis that RNA interference constrains L1 mobility seems
inherently reasonable: L1 mobility can be dangerous and L1 RNA,
the presumed target of RNAi, serves as a critical
retrotransposition intermediate. Despite its plausibility, proof
for this hypothesis has been difficult to obtain. Studies
attempting to link the L1 retrotransposition frequency to
alterations in RNAi activity have been hampered by the long times
required to measure retrotransposition frequency, the pleiotropic
and toxic effects of altering RNAi over similar time periods, and
the possibility that other cellular machinery may contribute to
the regulation of L1s. Another problem is that the commonly used
L1 reporter cassette may serve as a substrate for RNAi. Here we
review the L1-RNAi hypothesis and describe a genetic assay with a
modified reporter cassette that detects approximately 4 times more
L1 insertions than the conventional retrotransposition assay. RNAi SILENCING OF TRANSPOSABLE ELEMENTS RNAi is an evolutionarily conserved process of
sequence-specific posttranscriptional gene silencing (reviewed in
[ 1]). Double-stranded RNA (dsRNA) is cleaved by the
ribonuclease DICER into small interfering RNA species (siRNAs).
SiRNA molecules, in turn, target complementary RNA sequences for
destruction (reviewed in [ 2]). RNAi is postulated to play a
role in the silencing of transposable elements and viruses that
produce dsRNA [ 3, 4]. One line of evidence linking RNAi to
repressed transposition comes from the nematode, C
elegans [ 5, 6]. Tc1 elements, a class of DNA transposons, mobilize in somatic cells,
but are silenced in the germ line of C elegans. A number
of mutant C elegans strains that have lost this silencing
have also lost the ability to execute RNAi (though there were also
RNAi mutants that lacked this transposon mobilization phenotype)
[ 5]. The identification of specific genes, which when mutated
show activation of germline transposition, indicates that an
active transposon-silencing process exists in the germline
[ 5, 6]. Another line of evidence linking RNAi (or a mechanism
similar to RNAi) to the regulation of transposable elements
involves the I-factor in Drosophila. Mobilization of the
I-factor (an L1-like non-LTR retrotransposon) is regulated at
least in part by a homology-dependent silencing mechanism in the
female germline [ 7, 8]. This silencing mechanism has been
linked to a series of molecules that are implicated in the RNAi
pathway, including the Argonaute protein PIWI [ 9, 10]. By analogy, perhaps a sequence-dependent process of mobile element
silencing, such as RNAi, is used to regulate L1 mobility. As with
the above-mentioned examples, the regulation of L1 mobility may be
particularly relevant in the germline and in embryos. Mobility in
the germline or in embryos could result in inheritance of the new
insertion. These sites are also where L1s are believed to be most
active [ 11– 14]. Other mechanisms for recognizing and
responding to dsRNA, such as RNase L and PKR-mediated responses,
can cause apoptosis. While apoptosis seems like a reasonable
strategy for dealing with a wayward somatic cell, in the germline
or early embryo, apoptosis could be detrimental to the fitness of
the organism [ 14, 15]. Here we explore the thesis that the
mobility of human L1s is regulated by RNAi. L1 RETROTRANSPOSITION: HAZARDS AND CONSTRAINTS The human genome contains roughly half a million long interspersed
elements (L1s) that collectively account for 17% of its mass
[ 16]. Most new L1 insertions are “dead on arrival” due to
5′ truncation and nearly all but perhaps 60–100 L1 sequences in
the human genome are inactive due to truncation, inversion, or
mutation [ 17]. As discussed elsewhere in this issue, retrotransposition can be
hazardous because L1s can insert into genes, alter gene expression, shuffle exons, transduce 3′ flanking
sequences, mobilize Alu elements, and their replicative
mobilization adds significant DNA mass to the genome
[ 18– 24]. L1 insertions and recombination
events involving genomic L1 and Alu insertions have been reported
in a number of genetic disorders (reviewed in [ 25]). Although
it is possible that some functions of L1 are beneficial to mammals
(a most interesting recent demonstration involves the potential
role of L1s as diversity generators in the CNS, [ 26]), most
germline L1 insertions are likely to be neutral or negatively
selected. Negative selection of L1s is suggested by the higher
frequencies of full-length human L1 insertions on the sex
chromosomes than the autosomes (the former not being as able as
the latter to undergo purifying selection) and by the dominance
and limited periods of activity of single L1 subfamilies in some
primate lineages [ 27, 28]. L1 mobility in mammals appears to be actively constrained. An
indirect line of evidence for this constraint is that different
cell types exhibit different rates of retrotransposition, ranging
from 30% or higher in some transformed cell lines to fewer than one per million cells. In the mouse, the rate of germline
retrotransposition events using an L1-EGFP transgene is
approximately one event in 100 offspring [ 11, 13].
Analysis of L1 transcription, protein production and retrotransposition,
reveals different levels of L1 activity in different cell types,
with highest levels of activity noted in germ cells, embryonal
cells, and recently neuronal cells [ 11– 13, 26,
29, 30].
The factor(s) that assist L1 mobilization in some cell lines, but
not others, are not known. L1 RNA IS A LOGICAL TARGET FOR LIMITING L1 MOBILITY RNA is a logical target for cellular machinery to protect against
unwanted L1 proliferation. L1 RNA is required and may be
rate-limiting for retrotransposition. In cell-culture-based assays
with tagged human L1 elements, it has been shown that a decrease
in L1 mRNA leads to a decrease in L1 retrotransposition
frequencies, lending support to the idea that L1 activity can be
limited by regulating L1 transcript abundance [ 31,
32]. L1 RNA
is critical for retrotransposition because it encodes the
necessary ORF1 and ORF2 proteins, which act preferentially upon
the RNA that encoded them [ 33,
34]. This effect, termed
cis preference, may allow active L1s a greater
proliferative advantage than retroelements that mobilize in
trans because trans-mobilization can result in
the expansion of mutated rather than active elements. RNAi may be
able to counter this potential advantage of cis
preference by using nonfunctional L1 RNAs to inhibit functional
L1s. On the other hand, the high copy number of L1 insertions in
mammals may have been selected for L1s that are inefficiently
regulated by RNAi. If RNAi silences L1s, it does not do so with
perfect efficiency since L1 transcripts are detected, and some L1s
can still mobilize in the human genome. How (or even if) L1 RNA is recognized by cellular machinery is
unknown. If RNAi limits human L1 retrotransposition, the most
obvious possibility is that RNAi posttranscriptionally targets L1
mRNA. The presumed trigger for RNAi is double-stranded RNA
(dsRNA), although other forms of sequence-specific recognition or
unusual RNA secondary structure are possible. DsRNA has been
documented to be the target of RNAi-induced transposon silencing
in other species, most notably the Tc1 DNA transposon in C
elegans [ 5, 6].
Read-through transcription of dispersed Tc1
copies can form dsRNA as a result of “snap-back” of their
terminal inverted repeats (TIRs), which are complementary in
sequence. Human L1 retrotransposons are not flanked by
complementary TIRs, however there is considerable nucleotide
sequence similarity between active L1s [ 35]. This high level
of sequence similarity amongst active human L1 elements might
allow only a few L1 dsRNA molecules to silence many genomic L1s.
Sense and antisense L1 transcripts have been documented in human
teratocarcinoma cells [ 36]. There are two reports suggesting
the presence of long L1 dsRNA [ 37, 38], although thus far an
unequivocal demonstration of Dicer-derived L1 siRNAs or miRNAs
from mammalian cells has remained elusive [ 39– 41]. There are several ways in which L1 dsRNA could be
formed (see ). First, antisense L1 RNA could
arise as a read-through transcript from a heterologous promoter
element (). If sense and antisense
transcripts originating from different loci could form dsRNA, even
inactive copies of L1 could contribute to the loss of L1 mobility
(). As the genomic burden of L1 copies
increases, the level of L1 repression might also increase. On the
other hand, highly efficient silencing of all L1 copies in
trans could be problematic since L1s may influence the
human transcriptome significantly (reviewed in [ 20]). An
alternative is to selectively target L1 dsRNA that arises in
cis. In this connection, the L1 5′UTR has antisense
promoter activity at positions 400–600, providing an additional
source of antisense L1 RNA () [ 42,
43].
RNAi targeting the L1 5′UTR would be expected to selectively
restrain full-length (and therefore more likely to be active)
elements. Finally, L1 dsRNA can originate from the transcription
of inverted repeats (). A high copy number of
L1 sequences in mammalian genomes increases the likelihood of
generating inverted repeats either by genome rearrangements or by
insertions of L1 elements into or near themselves [ 44]. We
conducted a simple search for long inverted repeats
(> 200 bp) in the human genome, and a preliminary
analysis of several chromosomes indicates that the whole genome
contains tens of perfect inverted repeats of L1 sequences.
Transcription of such inverted repeats results in an efficient
dsRNA formation because this dsRNA folding is a first-order
reaction. Although the focus here is on dsRNA, there may be other
forms of L1 RNA that are recognized. L1 RNA species are
heterogeneous due to variable 5′ truncation, premature
polyadenylation, and inversion [ 45, 46]. HOW MIGHT RNAi SILENCE L1s? The most obvious possibility is that
RNAi limits L1 retrotransposition by decreasing the amount of L1
RNA. In this case, disruption of RNAi should increase L1 RNA
levels and result in an increased retrotransposition frequency.
The relationship between RNAi and repression of the LTR
retrotransposons MuERV-L and intracisternal A-particle (IAP) was
recently investigated in early mouse embryos [ 47]. Knocking
down DICER (with siRNA or dsRNA) resulted in a
50% increase in the abundance of MuERV-L and IAP transcripts
[ 47]. Recently, conditional dicer knock-out ES cells were
shown to exhibit slightly increased levels of IAP and L1
transcripts compared to dicer wild-type cells [ 48]. In
further support of this theory, L1 retrotransposons can form dsRNA
that is cleaved into siRNAs by DICER in cultured cells
[ 49, 50]. This analysis reveals that L1s can serve as targets
for RNAi, but does not address whether they do so in nature. Another possibility is that one or more components of the RNAi
machinery acts by silencing L1 insertions in chromatin via
methylation of L1 DNA [ 47]. Methylation has been proposed as
a genomic defense against transposable elements and may function
in an RNAi-dependent or independent manner to limit L1
transcription [ 53, 54]. Methylation of the L1 5′UTR has been
demonstrated in different cell types [ 55, 56]. Treatment of
3T3 cells with 5-azacytidine, a pyrimidine analog that inhibits
DNA methyltransferase, increases L1 transcript abundance
[ 57]. In mice, inactivation of methylases can result in
mobilization of retrotransposons including IAP elements and L1s
[ 58, 59]. On the other hand, methylation was not observed in
response to stable dsRNA expression in murine oocytes [ 47].
Consistent with the latter observation, a recent analysis in human
cancer cells suggests that RNAi-mediated transcriptional silencing
can arise independently of methylation [ 60]. Another nonmutually exclusive possibility is that RNAi
participates in altering chromatin accessibility. Heterochromatic
silencing and histone methylation have been tied to the RNAi
pathway in S pombe [ 61, 62]. Moreover, in the
filamentous fungus, Neurospora crassa, repression of the
L1-like retrotransposon Tad is dependent upon the
Argonaute protein QDE2 and DICER [ 63]. DNA and histone
methylation have also been implicated in transposon control in
Arabidopsis [ 64]. In certain yeast and plant
species, heterochromatin formation may be directed by siRNAs in an
Argonaute complex with similarities to the RNA-induced silencing
complex, suggesting that the processes of PTGS and transcriptional
gene silencing are intertwined [ 63]. Although plant L1-like
elements differ from mammalian L1 elements, a similar means of
mammalian L1-associated chromatin silencing may be at work. It is possible that RNAi acts upon L1s using all of these
pathways: degradation of L1 RNA (which limits the production of
new insertions), modification of L1 DNA sequences and chromatin
silencing (which should limit the activity of new or existing
functional L1s). The containment of L1s in regions of silenced
chromatin provides protection by suppressing their transcription,
mobility, and recombinational activity [ 65– 67]. ESTABLISHING A FUNCTIONAL LINK BETWEEN RNAi AND L1 RETROTRANSPOSITION Currently, the only direct evidence linking RNAi to the repression
of L1 elements in mammals is a slightly increased level of L1
transcripts in dicer deficient mouse ES cells [ 48]. Current
efforts to explore L1 regulation by RNAi in mammals are focused on
three areas: (i) demonstration of siRNAs derived from native L1
elements; (ii) determining whether L1 dsRNA is assembled in
cis (from the same L1) or if sense and antisense
transcripts originating from two different elements (assembly in
trans) can also trigger RNAi; and (iii) perturbing
components of the RNAi pathway and seeing if there are
corresponding alterations in the L1 retrotransposition frequency.
Exploration of the first area is under active investigation and is
discussed in detail elsewhere in this issue. Concerning the second
area, it has been assumed that dsRNA formation in trans
is minimal because xenogeneic L1s (eg, a human element in a mouse
cell) do not appear to be more active than syngeneic L1s (a human
element in a human cell) [ 32, 68]. However, the permissiveness
for L1 retrotransposition in these different cell types is not
controlled for. It is intriguing that L1 elements that have
genetically modified RNA sequences, but identical protein coding
sequences, can be far more active for retrotransposition
[ 32, 46]. While there are other potential reasons for this
(such as decreased premature polyadenylation and alterations in
RNA structure), it will be interesting to see if some of the
enhanced activity of synthetic L1s is due to different levels of
RNAi. The third area of investigation attempts to establish a
mechanistic link between RNAi and L1 retrotransposition. Analyzing L1 retrotransposition in cells with altered RNAi
activity is challenging. The first obstacle is to monitor the
mobilization of an active L1 in a sea of L1 sequences in the
genome. To get around this “needle-in-a-haystack” problem, L1
elements were tagged with antisense marker cassettes interrupted
in the sense direction by an intron [ 52]. These tagged
elements could then be monitored for retrotransposition by scoring
for expression of the marker (which could only occur after a cycle
of transcription, processing, reverse transcription, and
integration into a transcriptionally permissive region of the
genome, see ). Because the L1
retrotransposition construct contains sense and antisense
promoters, it may induce RNAi regardless of whether the L1 element
induces RNAi naturally. To circumvent the potential problem of having bidirectional
transcription in the L1-EGFP construct, we created a series of
EGFP-tagged L1 elements that lacked antisense promoter activity
(). Using a genetic assay to monitor
retrotransposition, our preliminary data reveal a 2-fold increase
in retrotransposition when constructs lacking the antisense
promoter in the EGFP marker were used, compared to the
conventional L1-EGFP construct ().
To control for length effects (better detection of
retrotransposition events due to a shorter marker cassette), we
created a construct with a “stuffer fragment” in place of the
EGFP promoter. This L1-stuffer construct also exhibited increased
retrotransposition compared to the standard L1-EGFP construct,
indicating that the basis for the increased retrotransposition
frequency was due to the absence of promoter activity rather than
being due to differences in marker length. Because the
retrotransposition frequency in this assay approaches 90%, we
may be underestimating the true retrotransposition frequency
(there may be more than one insertion per clone). The basis for
the increased retrotransposition activity of constructs lacking
the antisense promoter driving the EGFP marker cassette is
unresolved. Perhaps these constructs will be helpful in future
studies that attempt to link RNAi to the regulation of L1
retrotransposition. ALTERNATIVE SILENCING PATHWAYS MEDIATED BY DsRNA DsRNA can induce several different pathways in mammals. One of
them is RNA editing, a process in which
adenosines are converted to inosine in nuclear dsRNA by the enzyme
adenosine deaminase (ADAR). Editing of dsRNA can
occur in a site-selective or promiscuous fashion. The latter
results in the generation of a series of variably mutated RNA
species. DsRNA longer than 50 bp in which > 20% A-to-I
editing has occurred is referred to as hyperedited [ 69].
Based largely on work with polyoma virus, hyperedited RNA may be
retained and/or sequestered in the nucleus [ 69]. L1 RNA can serve as a substrate for RNA editing [ 70, 71].
However, the effects of RNA editing on L1 activity are unknown.
Since RNA editing affects dsRNA without targeting homologous
copies of single-stranded RNA, editing may have a smaller impact
on L1 retrotransposition than RNAi. If L1 RNA editing is similar
to Alu editing, most RNA duplexes would be formed intramolecularly
due to base pairing between two oppositely oriented Alus residing
in the same RNA molecule [ 72]. Such duplexes would be
expected to have imperfect base pairing between neighboring
oppositely oriented L1 elements and could promote editing rather
than RNAi. RNA editing may further help L1 to evade RNAi because
hyperedited L1 dsRNA would be probably processed less efficiently
into siRNAs and such siRNAs would not base pair as well with their
targets. This idea is consistent with the observation that RNAi is
antagonized by hyperediting [ 73] and that the phenotype of
ADAR mutants can be rescued by mutations in RNAi [ 74]. In addition to siRNA and RNA editing, longer L1 dsRNA molecules
can induce additional cellular responses [ 75]. Longer dsRNA
molecules can be recognized by the dsRNA-dependent protein kinase
PKR, which, when activated, results in interferon-mediated
activation of the Jak-Stat pathway and cellular upregulation of
interferon-regulated genes [ 76]. This mechanism of cytokine
defense is an innate immune response that likely arose to combat
viruses, which frequently produce dsRNA. Activation of PKR by
dsRNA results in its autophosphorylation and subsequent
phosphorylation of the eukaryotic initiation factor 2α (eIF2α), causing general inhibition of cellular protein
synthesis [ 76]. Another pathway of dsRNA regulation involves
RNaseL, a potent riboendonuclease.
RNaseL can be indirectly triggered by dsRNA through an increase in
2′–5′ oligoadenylates. 2′–5′ oligoadenylates are produced
from ATP by 2′–5′ oligoadenylate synthetases, which are
activated by dsRNA [ 77]. In addition to the nucleolytic
properties of RNaseL, the enzyme also upregulates type I
interferon genes by sequestering NFκB transcription factors [ 77]. Certain cell types, for example those of myeloid origin,
constitutively express receptors that recognize dsRNA [ 78].
The toll-like receptor (TLR)3 recognizes and binds to dsRNA
[ 79]. TLR3 is expressed on the cell surface as well as in
intracellular vesicles [ 80]. Thus, dsRNA can be recognized by
TLR3 internally, as an intermediate in viral replication, or
externally, as dsRNA leaks from dying cells [ 78]. Recognition
of dsRNA by TLR3 initiates the binding of NFκB and IRF-3
transcription factors to the promoters of type I interferon genes
leading to their upregulation, which can eventually cause cell
death via apoptosis [ 80]. Mammalian oocytes, embryos, and embryonic stem cells do not induce
a dsRNA-mediated interferon response, but utilize the RNAi pathway
to respond to long dsRNA [ 14, 15,
81]. In contrast, somatic
cells might be more likely to use an interferon pathway when confronted
with long dsRNA species [ 82]. The rationale for using
different dsRNA recognition pathways in progenitor cells versus
somatic cells is that embryos may not be able to afford the luxury
of shutting down individual cells if trouble arises. In contrast,
adult mammalian cells can apoptose with little to no effect on the
organism as a whole. It may be that the secondary products of L1
dsRNA dicer-mediated endonucleolytic cleavage activate the
PKR-interferon pathway in adult differentiated cells, which
induces cell death. L1 retrotransposons have shaped the mammalian genome and
contribute significantly to its mass, yet their mobility appears
to be actively constrained. Along with other cellular defense
mechanisms, RNAi may participate in cell-type-specific,
multifaceted defense against L1 mobility that includes RNA
destruction, DNA methylation, and heterochromatin formation. The
development of new genetic models of RNAi deficiency in mammals,
coupled with a genetic assay for monitoring L1 retrotransposition
events, may help to advance our understanding of how L1 mobility
is regulated. We thank Richard Schultz, Greg Hannon, and members of the Luning
Prak Lab for helpful discussions and we gratefully acknowledge
Sarah Fox and Janet Sallit for skilled technical assistance. This
work was supported by NIH R01 CA108812 to Eline T Luning
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