A potential role for RNA interference in controlling
the activity of the human LINE-1 retrotransposon
Hope, 1450 East Duarte Road, Duarte, CA 91010-3011, USA and3Division of Molecular Genetics, Integrated DNA
Technologies Inc., 1710 Commercial Park, Coralville, IA 53341-2760, USA
Received November 16, 2004; Revised December 16, 2004; Accepted January 14, 2004
Long interspersed nuclear elements (LINE-1 or L1)
comprise 17% of the human genome, although
only 80–100 L1s are considered retrotransposition-
competent (RC-L1). Despite their small number,
RC-L1s are still potential hazards to genome integrity
through insertional mutagenesis, unequal recombi-
nation and chromosome rearrangements. In this
study, we provide several lines of evidence that the
LINE-1 retrotransposon is susceptible to RNA inter-
ference (RNAi). First, double-stranded RNA (dsRNA)
generated in vitro from an L1 template is converted
into functional short interfering RNA (siRNA) by
DICER, the RNase III enzyme that initiates RNAi in
human cells. Second, pooled siRNA from in vitro
cleavage of L1 dsRNA, as well as synthetic L1
siRNA, targeting the 50-UTR leads to sequence-
retrotransposition from a highly active RC-L1 clone
in cell culture assay. Our report is the first to demon-
strate that a human transposable element is sub-
jected to RNAi.
The majority of the human genome comprises DNA from
repetitive sequences and mobile genetic elements. Retro-
transposons, mobile genetic elements that move through
an RNA intermediate in a process termed retrotransposition,
are the most abundant mobile elements and comprise ?40%
of the human genomic DNA (1). Of these retrotransposons,
the non-long terminal repeat (non-LTR) long interspersed
nuclear elements (LINEs or L1) are referred to as autono-
mous retrotransposons because L1s encode their own pro-
teins necessary for retrotransposition (2). Although over 99%
of L1 sequences are inactive, either through deleterious
mutations, 50end truncations, or through internal rearrange-
retrotransposition-competent LINE-1 elements (RC-L1s).
The consensus RC-L1 is 6 kb in size and contains a 50-
untranslated region (50-UTR) with an internal promoter,
two non-overlapping open reading frames (ORF1 and
ORF2) and a 30-UTR that terminates with a poly(A) tail
(2). ORF1 encodes a 40 kDa (p40) RNA binding protein
that forms ribonucleoprotein particles (RNPs) with L1 RNA
(3–5). ORF2 produces a 150 kDa protein with an N-terminal
endonuclease and a C-terminal reverse transcriptase domain
(6,7). A unique feature of L1s is that the two L1 ORF pro-
ducts act preferentially on the RNA that encodes them, a
characteristic known as cis-preference, which might prevent
rampant retrotransposition of other non-autonomous RNAs
from disrupting the genome (8,9).
Despite the small number of RC-L1s, and the constraints
placed upon their movement by cis-preference, a number of
disease-causing mutations are attributed to insertional muta-
genesis byL1 (2,10–12).
mutagenesis, unequal recombination between dispersed L1s
and gene sequences is associated with several disease etiolo-
gies and has the potential to cause even more genomic damage
due to the large number of L1 sequences (>500 000) that
populate the human genome (13–15). Moreover, characteriza-
tion of retrotransposition events using tagged RC-L1 clones in
cultured cells indicate that ?10% of L1 insertions are accom-
panied by large chromosomal rearrangements, suggesting that
active L1s could also lead to genomic instability (16,17).
In other eukaryotes, the RNA interference (RNAi) pathway
suppresses the activity of transposable elements that pre-
dominate lower eukaryotic genomes. For example, RNAi
is involved in silencing transposons in the germ line of
Trypanosoma (18–22). Furthermore, disruption of the RNAi
function in eight-cell stage mouse embryos results in
transcripts (23). It is likely, therefore, that RNAi represents
Inaddition to insertional
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Nucleic Acids Research, 2005, Vol. 33, No. 3
an evolutionary important mechanism whereby eukaryotic
genomes are protected from the threat posed by mobile genetic
RNAi is a conserved eukaryotic mechanism in which the
double-stranded RNA (dsRNA) recognizes homologous
mRNAs and causes sequence-specific degradation in a
multi-step process. RNAi is initiated by the cleavage of
endogenous long dsRNA or short hairpin RNA (shRNA)
by the RNase III enzyme DICER into 21–23 nt small inter-
fering RNA (siRNA) effector molecules (24–28). These
siRNAs recognize their cognate mRNA and become asso-
ciated with a large multi-protein complex referred to as the
RNA-induced silencing complex (RISC) that destroys target
mRNAs by endonucleolytic cleavage at regions homologous
to the siRNA (29,30). Although only a few distinct compo-
nents of the RNAi pathway are known, such as DICER,
genetic and biochemical studies link members of the Argo-
nautefamily tothe RISC complex (29,31,32). In addition, only
a few endogenous siRNAs have been identified along with
their targets. However, using both genetic and biochemical
approaches several siRNAs derived from mobile genetic
elements such as the Tc1 transposon in C.elegans and the
Ingi retroposon in Trypanosoma are known to exist (18,22),
although thus far there have not been any demonstrations
of endogenous siRNAs targeting transposable elements in
In the present study, we show that dsRNA transcribed
from a human L1 retrotransposon template is a substrate for
in vitro cleavage by the RNase III enzyme DICER, and that the
resulting L1 siRNA leads to RNAi-mediated degradation of
a hybrid L1 fusion transcript. Moreover, both synthetic and
in vitro processed L1 siRNA can inhibit L1 retrotransposon
activity in an established cell culture retrotransposition
assay. These results provide proof of principle that the
human RNAi machinery can act on L1 transcripts and limit
MATERIALS AND METHODS
HeLa (CCL-2) and HCT116 (CCL-247) cells were obtained
from ATCC. HeLa cells were grown in DMEM (Invitrogen,
Carlsbad, CA) supplemented with 10% fetal bovine serum
(FBS), 20 U/ml penicillin/streptomycin and 2 mM
glutamine. HCT116 cells were grown in McCoy’s 5A medium
(Irvine Scientific, Irvine, CA) supplemented with 10% FBS,
20 U/ml penicillin/streptomycin and 1.5 mM L-glutamine.
Both cell lines were cultured in a humidified 5% CO2incu-
bator at 37?C and split when they reached confluence.
Luciferase reporter vectors. Plasmid pGL3U1 was cloned by
PCR amplification of the human U1 promoter and inserting it
upstream of the firefly (FF) luciferase coding sequence in the
pGL3 basic vector (Promega, Madison, WI). The wild-type
50-UTR containing the L1 internal promoter was PCR ampli-
fied from either pCEP4/L1.3neo-ColE1 or pCEP4-L1RPneo
using Pfu DNA polymerase (Stratagene, La Jolla, CA) and
primers (HindIII-L1-F/HindIII-L1-R, Table 1). The resulting
PCR products were digested with HindIII and subcloned into
the pGL3 basic vector (Promega, Madison, WI), upstream
of the luciferase cDNA to generate pHSS101/L1.3 and
pHSS101/RP, respectively. The correct sequence of each luci-
ferase construct was confirmed by DNA sequencing atthe City
of Hope DNA Sequencing Core.
Retrotransposon and expression vectors. Plasmids pCEP4-
L1RPneo (wild type) and pCEP4-L1.3-JM111 (mutant) are
LINE-1 constructs that contain the mneoI retrotransposition
indicator cassette described previously and were generously
provided by Drs Haig H. Kazazian and John V. Moran
(33,34). Plasmid pEMD-GFP is a mammalian reporter vector
expressing the green fluorescent protein (Packard Instrument
Table 1. Primers and synthetic siRNAs used in this study
L1 50-UTR dsRNA
L1 ORF1 dsRNA
L1 ORF2A dsRNA
L1 ORF2B dsRNA
L1 50-UTR siRNA #749
Firefly luciferase siRNA
HIV Rev siRNA
All primers and siRNA are listed 50!30.
Nucleic Acids Research, 2005, Vol. 33, No. 3847
Madison, WI) is a mammalian expression vector that contains
an SV40-driven neomycin phosphotransferase gene allowing
for stable selection of transfected cells.
Meriden, CT).PlasmidpCIneo (Promega,
Generation of dsRNA. cDNA templates with opposing T7
phage RNA polymerase promoters were generated by PCR
by incorporating the T7 sequence into both forward and
reverse primers. A complete list of primers used to produce
cDNA templates for in vitro transcription is given in Table 1.
PCR products were amplified from the pCEP4-L1RPneo
plasmid template using Pfu DNA polymerase and then gel
extracted following agarose gel electrophoresis. Additional
long dsRNA was generated from a b-galactosidase template
(plasmid pCH110, Pharmacia, Piscataway, NJ) which served
as a negative control in RNAi experiments. These cDNA
templates were subjected to in vitro transcription using the
Megascript T7 in vitro transcription kit (Ambion, Austin, TX)
according to the manufacturer’s directions. The resulting long
dsRNA was heated to 75?C for 5 min in a heat block and
allowed to cool slowly at room temperature to anneal the
opposing RNA strands. Annealed dsRNA was purified using
the RNAid kit (Bio101, Vista, CA) and examined for quality
on a 2.5–3% agarose gel.
Diced siRNA production. About 60 mg of dsRNA was incu-
bated with recombinant human DICER (Block-it Dicer kit,
Invitrogen, Carlsbad, CA) as recommended by the manufac-
turer. The resulting siRNA was purified using siRNA purifica-
tion columns (Block-it Dicer kit) and the quality and quantity
Synthetic siRNA. Synthetic siRNA was designed using a web-
based algorithm found at http://proteas.uio.no/siRNAbeta.
html (35). Briefly, the nucleotide sequences of the L1
50-UTR and ORF1 regions from the RC-L1 clone L1RP
were used to generate a list of siRNA candidates. Each can-
didate was then queried against the RefSeq database and the
siRNAs that exhibited limited sequence homology to known
mRNAs (<15/19 nt), especially at the 50end of the antisense
strand, were chosen for synthesis. Individual siRNA strands
were synthesized chemically (IDT, Coralville, IA) as 21mers
with dTdT 30overhangs. Sense and antisense siRNA strands
were annealed by combining equimolar amounts, heating to
75?C for 5 min and then cooling slowly to room temperature.
In addition, synthetic siRNAs targeting the FF luciferase
coding region and HIV Rev transcript were also produced
(IDT, Coralville, IA). The sequence of L1 50-UTR #749,
FF luciferase and HIV Rev synthetic siRNAs are listed
in Table 1.
HCT116 (colon carcinoma) were seeded in 24-well culture
dishes and transfected at 70–80% confluence with Lipofecta-
mine 2000 according to the manufacturer’s recommenda-
tions (Invitrogen, Carlsbad, CA). Each well received 1 ng
of the Renilla luciferase expression vector phRL (Promega,
Madison, WI), 10 ng pEMD-GFP, 200 ng luciferase reporter
vector (either pGL3U1 or pHSS101 derivatives) and 0–250 ng
(Stratagene, La Jolla, CA) was added so that each well
received a total of 800 ng of nucleic acid. All samples were
transfected in duplicate or triplicate and the experiment was
performed a minimum of three times. For control transfec-
tions, either synthetic siRNA targeting the FF luciferase trans-
gene (positive control for the RNAi effect), synthetic siRNA
targeting the HIV Rev transcript (negative control) or negative
control diced siRNA targeting the b-galactosidase mRNA
was included instead of siRNA produced from L1 dsRNA.
The transfections were performed as follows: first, the
serum-containing media was removed and the cell monolayer
was incubated with 100 ml of the transfection mix and returned
to the incubator. After 1 h, the transfection mix was removed
and 500 ml of serum-containing media was added to the mono-
layer and the cells were returned to the 37?C incubator. Luci-
ferase assays were performed 24 h after transfection using the
Dual Luciferase Assay system according the manufacturer
(Promega, Madison, WI).
siRNA. Inaddition, pBluescript IISK+
L1 retrotransposition experiments
L1RPneo were performed essentially as described previously
(36,37). HCT116 or HeLa cells were seeded in 6-well plates
and transfected with Lipofectamine 2000 when they reached
70–80% confluence. Each well received 1 mg of RC-L1 con-
struct, 100 ng of plasmid pEMD-GFP to normalize transfec-
tion efficiency, 0–250 ng siRNA and pBluescript II SK+ up to
2 mg of total nucleic acid. Transfections were performed in the
absence of serum similar to the luciferase assay transfections.
At 24 h post-transfection, each well was trypsinized, counted
with a hemocytometer and serial dilutions were plated onto
100 mm dishes. The remaining cells were subjected to flow
cytometry to normalize for transfection efficiency between
samples. At 36 h post-transfection, the serially plated cells
were selected using G418 at a concentration of 400 mg/ml
(HCT116) and 600 mg/ml G418 (HeLa).
RT–PCR and genomic DNA PCR
HCT116 cells 24 h post-transfection and treated two times
with RNAse-free DNase I to remove any contaminating geno-
mic and plasmid DNA. About 3 mg of RNA was used for
(Invitrogen, Carlsbad, CA) and random hexamers to prime
first-strand synthesis. Parallel cDNA reactions were carried
out for each sample, except M-MLV reverse transcriptase
was omitted from the reaction. Separate PCR reactions for
GAPDH and FF luciferase transcripts were carried out in a
total volume of 50 ml containing 1· Taq buffer, 1.5 mM
MgCl2, 0.2 mM of each dNTP, 0.2 mM of each primer,
2.5 U Taq DNA polymerase (Brinkmann Instruments, Inc.,
Westbury NY) and 10 ml (?100 ng) of the appropriate
cDNA. The PCR was carried out at 95?C for 5 min
(1 cycle) followed by 94?C for 30 s, 55?C for 30 s and
72?C for 30 s. Primers used to amplify GAPDH and FF luci-
ferase transcripts are listed in Table 1. PCR cycles for GAPDH
and FF luciferase were 22 and 24, respectively, as determined
by cycle study analysis to determine the linear range of ampli-
fication. PCR products were separated on a 1.5% agarose gel
848Nucleic Acids Research, 2005, Vol. 33, No. 3
and visualized by ethidium bromide staining. The amplified
products were quantified using an Alpha-innotech imager and
Genomic DNA PCR. Genomic DNA was harvested from
HCT116 G418Rclones using the QiaAmp DNA Blood Kit
(Qiagen, Valencia, CA). Each PCR reaction was performed in
a 50 ml vol containing 1· Taq buffer, 1.5 mM MgCl2, 10 U of
Taq polymerase, 0.2 mM dNTPs, ?100 ng of genomic DNA
template and 200 ng of primers 437S and 1808AS (Table 1),
which are specific for the neoRgene. PCR was performed
using a cycling program of 95?C, 5 min (1 cycle); 94?C,
1 min; 64?C, 30 s; 72?C, 1 min (30 cycles); a final extension
of 72?C for 10 min. One-tenth of the PCR reaction was loaded
onto a 1.5% agarose gel and visualized by ethidium bromide
Recombinant human DICER can convert L1 dsRNA
To investigate whether the human RNAi machinery has the
potential to limit the activity of the human LINE-1 (L1) non-
LTR retrotransposon, we first determined if long dsRNA
generated from the L1s 50-UTR, ORF1 and ORF2 regions,
could be converted into siRNA by recombinant human
DICER (Figure 1A). As shown in Figure 1B, L1 dsRNA
was converted to siRNA of the appropriate size in an efficient
and processive manner. Almost all of the long L1 dsRNA was
gene silencing experiments. However, before the gene silen-
cing experiments could begin, the pool of siRNA needed to
be purified from any contaminating long dsRNA because of
the likelihood that the transfection of contaminating dsRNA
would lead to non-specific reduction of mRNA and translation
inhibition through interferon induction. Weexperimented with
a number of ways to separate the siRNA from residual long
dsRNA, including purification from both PAGE and agarose
gels following electrophoresis. However, we found that using
size-exclusionspin columns was the mostefficient method and
produced the highest quantity of siRNA, free of contaminating
long dsRNA, as determined by agarose gel electrophoresis.
The diced L1 siRNA is functional at targeting
a 50-UTR-driven hybrid reporter transcript
Next, we assessed whether L1 siRNA was capable of
RNAi-mediated gene silencing of homologous L1 sequences.
Forthispurpose,weclonedthe50-UTRfromthe RC-L1 clones
L1RPand L1.3 upstream of the FF luciferase reporter gene, to
generate pHSS101/RP and pHSSS101/L1.3, respectively. The
L1 50-UTR is ?910 nt and contains an internal promoter with
transcription beginning at or near the first nucleotide of the
element (38,39). Thus, the 50-UTR-driven luciferase construct
produces a hybrid mRNA with L1 sequence at the 50end of the
transcript followed by the luciferase coding region and a poly
(A) tail. Diced 50-UTR L1RPsiRNA, along with the respective
pHSS101 construct, was co-transfected into HCT116 and the
FF luciferase activity was determined after 24 h. A limiting
amount of Renilla luciferase was included in each transfection
to normalize for transfection efficiency between wells. No
differences in Renilla luciferase activity were observed
between different siRNA treatments, suggesting that the
diced 50-UTR siRNA did not produce any measurable off-
target effects (data not shown).
Figure 1. L1 dsRNA is a substrate for recombinant human DICER. (A) Schematic representation of a retrotransposition-competent LINE-1 (RC-L1) illustrating
the 50-UTR with internal promoter, ORF1 product p40, inter-ORF region (bold black line), ORF2 domains EN (endonuclease) and RT (reverse transcriptase), and
dsRNA; closed arrowhead, L1 siRNA, measuring ?21–23 nt.
Nucleic Acids Research, 2005, Vol. 33, No. 3849
In the presence of 50 ng diced 50-UTR L1RPsiRNA, the FF
luciferase activity was reduced by 62% (P < 0.001), indicating
that diced L1 siRNA could efficiently inhibit expression of the
hybrid 50-UTR-luciferase transcript. L1 siRNA inhibited the
expression ofthe hybrid transcriptina dose-dependent manner
and reached a maximum of 75% inhibition with 100 ng diced
50-UTR siRNA (P = 0.01) (Figure 2). In addition to targeting
the L1RP50-UTR from which the dsRNA template for in vitro
dicing was generated, we investigated whether L1RP50-UTR
siRNA could inhibit the expression of the hybrid transcript
Figure 2. L1 siRNA specifically inhibits the expression of a hybrid luciferase transcript driven by the L1 50-UTR internal promoter. HCT116 cells were co-
transfected with the indicated firefly (FF) luciferase expression vector and increasingamounts of L1 siRNAor control siRNA.Relative FF luciferase activity in the
absence of siRNA was set at 1.0. The amount of each specific siRNA is indicated below the graphs. (A) Luciferase assays performed with in vitro ‘diced’ siRNA.
targeting nucleotides 749–769 of the L1 50-UTR. Irrelevant synthetic siRNA targeting the HIV Rev transcript was included as the negative control.
850 Nucleic Acids Research, 2005, Vol. 33, No. 3
driven by the 50-UTR from another RC-L1 clone, L1.3.
Because the L1RP50-UTR and L1.3 50-UTR exhibit greater
than 98% sequence identity, we suspected that L1RP-derived
50-UTR siRNA could reduce luciferase expression of the
L1.3-driven hybrid transcript (40). As expected, L1RP-derived
50-UTR siRNA inhibited the FF luciferase activity from a
construct driven by the L1.3 50-UTR by more than 51%
(P = 0.002) (Figure 2). This amount of inhibition of the
L1.3-driven transcript is significantly less (P = 0.001) than
the 62% inhibition measured for the L1RP-driven transcript.
To rule out the possibility that the lower inhibition of the
L1.3-driven transcript is due to increased promoter activity
of L1.3 compared with L1RP, we calculated the promoter
strength of each 50-UTR-driven vector in the absence of
siRNA. Following normalization to Renilla luciferase, tran-
scriptionfromthe L1RP50-UTRinternal promoterwas 1.4-fold
higher (P = 0.009, data not shown) than the expression of FF
luciferase from the L1.3 50-UTR. Thus, the increased 50-UTR
siRNA-mediated inhibition of the L1RP-driven transcript
may reflect the perfect complementarity of the L1RP-derived
siRNA, whereas the L1.3-driven hybrid mRNA is predicted
to contain small sequence differences at the 50end.
Because the siRNA used in these experiments represents a
pool derived from in vitro dicing of long 50-UTR dsRNA, we
wished to exclude the possibility that the observed inhibition
in luciferase activity was due to the 50-UTR siRNA acting
against the luciferase region of the hybrid transcript. Thus,
we co-transfected the L1RP50-UTR siRNA with a separate FF
luciferase expression construct driven by the human U1 pro-
moter. This U1-driven luciferase transcript contains no sig-
nificant homology to the L1RPsiRNA. As shown in Figure 2A,
no significant decrease (P = 0.404) in luciferase activity was
observed for the U1-driven luciferase construct in the presence
of 100 ng L1RP50-UTR siRNA. However, synthetic luciferase
siRNA significantly reduced expression from the U1-driven
construct by >74%, indicating that this transcript is also sus-
ceptible to RNAi-mediated inhibition (data not shown). As an
additional control for non-specific inhibition by diced siRNA,
we ‘diced’ long dsRNA derived from the b-galactosidase
coding region and co-transfected this b-gal siRNA with both
pHSS101/L1.3 and pHSS101/L1RP. The diced b-gal siRNA
showed no reduction of FF luciferase activity for both the
L1RP- (P = 0.903) and L1.3-driven (P = 0.103) hybrid tran-
scripts (Figure 2A). Thus, the decrease in luciferase activity
mediated by L1 siRNA was specific to the hybrid transcript,
and not to the luciferase sequence itself.
To conclusively demonstrate that the L1 50-UTR is suscep-
tible to RNAi, we designed three synthetic siRNAs against
different regions of the 50-UTR (50-UTR #319, #454, #749,
respectively). We carefully selected siRNA sequences that
showed no significant homology to endogenous mRNAs by
querying the RefSeq database. In preliminary co-transfection
experiments with pHSS101/RP, 50-UTR #749 siRNA was
determined to be the most effective at silencing the hybrid
transcript and chosen for additional experiments (data not
shown). In transient co-transfection experiments with both
pHSS101/RP and pHSS101/L1.3, as little as 10 ng of 50-UTR
#749 could decrease the luciferase activity by >75%, reaching
a maximum suppression of 85% at 50 ng of the synthetic
50-UTR #749 (Figure 2B). We did not observe increased silen-
cing of luciferase activity at siRNA levels >50 ng, suggesting
that the RNAi pathway is saturated at higher amounts of
50-UTR #749. Irrelevant siRNA targeting the HIV Rev
mRNA produced no inhibition of both 50-UTR-driven
The diced siRNA reduces luciferase activity by
decreasing mRNA levels of the 50-UTR-driven transcript
To confirm that the observed decrease in FF luciferase activity
was due to 50-UTR siRNA targeting the hybrid transcript
for RNAi-mediated degradation, we measured the expression
of the remaining hybrid mRNA levels by semi-quantitative
RT–PCR. As shown in Figure 3, the 50-UTR siRNA reduced
the hybrid transcript levels by 56% compared with that in the
absence of siRNA. In addition, synthetic FF luciferase siRNA
reduced the hybrid transcript levels by 64%. Thus, the pool of
50-UTR siRNA is acting on the hybrid transcript by targeting
it for degradation, presumably through an RNAi-mediated
mechanism, and the reduced luciferase activity observed in
the presence of 50-UTR siRNA is not due to translational
inhibition. While we measured a 56% reduction in hybrid
mRNA levels in response to 50-UTR siRNA, we observed a
greater reduction in luciferase activity from the same sample
using the commercial dual luciferase assay. This discrepancy
most likely arises from the short half-life of luciferase mRNAs
or the decreased sensitivity of semi-quantitative RT–PCR
compared with the highly sensitive luciferase assay.
Figure 3. L1 siRNA inhibits luciferase expression through RNAi-mediated
degradation of the hybrid transcript. (A) RNA harvested from HCT116 cells
co-transfected with pHSS101/RP and the indicated siRNA was subjected
to semi-quantitative RT–PCR analysis with primers set to amplify the FF
luciferase transgene and a separate set of primers to amplify the GAPDH gene
as an internal loading control. FF luciferase expression was normalized to
GAPDH levels. Relative luciferase expression in the absence of siRNA was
set at 1.0. (B) Graphical representation of the semi-quantitative RT–PCR data.
Columns represent mean FF luciferase expression (–standard deviations) of
three independent PCR experiments.
Nucleic Acids Research, 2005, Vol. 33, No. 3851
L1 siRNA can reduce the activity of an RC-L1 in a
transient retrotransposition assay
Next, we investigated whether L1 retrotransposition activity is
reduced in the presence of siRNA homologous to different
regions of an RC-L1 transcript by employing an established
transient retrotransposition assay. In this assay, a tagged
RC-L1 expression construct containing an indicator cassette
(mneoI) is transiently co-transfected with a limiting amount of
a GFP expression vector to monitor transfection efficiency.
The mneoI retrotransposition indicator cassette consists of
the neomycin phosphotransferase (neoR) selectable marker,
a heterologous promoter and polyadenylation signal and is
cloned in the 30-UTR in the antisense orientation with respect
to the L1 transcript.Inaddition, the neoRgene isinterruptedby
intron 2 of the g-globin gene, which is in the sense orientation.
Thus, expression of the mneoI cassette will occur only if
spliced, reverse transcribed and integrated into chromosomal
DNA. Expression of the neoRgene from the heterologous
promoter will then give rise to G418 resistance (42). We
chose the RC-L1 clone L1RPbecause it is one of the most
active RC-L1 clones examined to date and would not only
provide a high baseline level of retrotransposition from which
to observe a decrease due to siRNA targeting the L1 transcript,
but would also afford a good measurement of the potential for
RNAi to limit L1 activity (34).
Before proceeding with the retrotransposition assays, we
performed preliminary experiments to ensure that the diced
L1 siRNA did not produce any off target effects which would
pose problems during G418 selection, such as targeting the
neoRmRNA. Therefore, we co-transfected HCT116 cells with
the plasmid pCIneo, a mammalian expression vector that
contains an SV40-driven neoRselectable marker, and diced
siRNA from the L1s 50-UTR, ORF1, or irrelevant LacZ
siRNA. Serial dilutions of cells were plated and G418 selec-
tion was carried out 36 h after transfection for 14 days. No
difference in stable G418 selection was observed for any of the
diced siRNAs relative to pCIneo transfected without siRNA,
demonstrating that the diced L1 and irrelevant LacZ siRNA
do not produce non-specific inhibition (data not shown).
Next, we transfected HCT116 and HeLa cells with the
tagged RC-L1RPconstruct alone or with serial dilutions of
siRNA targeting the 50-UTR. In the presence of as little as
50 ng diced 50-UTR siRNA, the retrotransposition efficiency
in HCT116 cells was significantly reduced by 75% relative to
the no siRNA control (P < 0.001). The decrease in retro-
transposition was dose-dependent and reached a maximum
reduction (91%, P = 0.004) with 250 ng of diced 50-UTR
siRNA (Figure 4A). Although we measured a small 15%
decrease in the retrotransposition activity with 50 ng of
diced LacZ siRNA (P = 0.02), this difference may be the result
of short regions of homology between the diced LacZ and
both the 50-UTR and ORF2 regions of L1RP. Retrotransposi-
tion activity was almost completely abolished with 250 ng
synthetic 50-UTR #749 siRNA (>99%, P < 0.001). This
large decrease was specific to the 50-UTR, as we did not detect
any decrease in the retrotransposition activity in HCT116
cells in the presence of 250 ng synthetic irrelevant siRNA
targeting the HIV Rev transcript (P = 0.92). We observed
a more pronounced reduction in the retrotransposition
frequency in HeLa cells transfected with both 50 ng (84%,
P = 0.02) and 250 ng (94%, P = 0.009) of the diced 50-UTR
siRNA relative to the no siRNA control (Figure 4B). Again,
irrelevant HIV Rev siRNA produced no significant reduction
in L1 activity (P = 0.16).
To further study the potential of RNAi to limit the activity
of the L1 retrotransposon, we also measured retrotransposition
activity in HCT116 cells exposed to diced ORF1 siRNA.
ORF1 siRNA also reduced retrotransposition activity in
a dose-dependent manner relative to the no siRNA control
(Figure 4A). The observed reduction in response to 50 ng
diced ORF1 siRNA was significantly greater than the reduc-
tion in the retrotransposition activity measured with the
same concentration of diced 50-UTR siRNA (75% versus
96%, P < 0.02). We were unable to design synthetic ORF1
siRNA using the web-based algorithm that lacked significant
homology to known endogenous mRNAs after querying the
RefSeq database. To confirm that the G418 resistance in
HCT116 clones was derived from a retrotransposition event,
proper splicing of the g-globin intron disrupting the neoRgene
was confirmed by PCR analysis of genomic DNA from several
stable clones (data not shown).
We have several lines of evidence providing proof of principle
that the RNAimachinery can act on L1s and limit their activity
in a transient retrotransposition assay. First, long dsRNA tran-
scribed in vitro from an RC-L1 clone is efficiently processed
into functional siRNA by the RNase III enzyme DICER.
Second, both synthetic and in vitro ‘diced’ L1 siRNA inhibit
the expression of an L1 hybrid transcript by RNAi-mediated
degradation. Third, siRNA targeting different regions of the
L1 can limit the activity of an RC-L1 in a cultured cell retro-
We initially decided to target the 50-UTR of the L1 retro-
transposon because this region is presumably part of most,
if not all, remaining RC-L1s that populate the human genome.
In addition, the L1 50-UTR contains both a sense promoter,
as well as an antisense promoter (ASP), and thus might play
a larger role in the transcriptional regulation (43) through the
production of L1 dsRNA. Furthermore, because the 50-UTR
contains the L1s internal promoter with transcription begin-
ning at or near the first nucleotide of the L1 at an unconven-
tional start site (50-GGGGG-30), we reasoned that it would
be easy to test the efficacy of siRNAs targeting the 50-UTR
by creating a fusion construct with a measurable reporter
gene such as FF luciferase (38,39). Indeed, we found that
the 50-UTR-driven reporter construct achieved high levels
of luciferase expression in both HCT116 and HeLa cells,
consistent with the data from other studies examining the
promoter activity of the L1 50-UTR, suggesting that the factors
necessary for efficient transcription are present in many
somatic cells types (38,39,44,45).Even with the robustexpres-
sion of the 50-UTR driven constructs, we observed a sharp
decrease in the luciferase activity from the hybrid transcript
in the presence of either in vitro ‘diced’ or synthetic siRNA
targeting the 50-UTR. The decrease in the luciferase activity
was not due to non-specific targeting of the luciferase coding
region,as acontrolconstructexpressing FFluciferasefrom the
852Nucleic Acids Research, 2005, Vol. 33, No. 3
Figure 4. L1 siRNA can limit the activity of the RC-L1 clone, L1RP, in a transient retrotransposition assay. HCT116 or HeLa cells were transfected with
pCEP4-L1RPneo alone or co-transfected with different siRNA. The retrotransposition activity was determined using a transient retrotransposition assay follow-
ing G418 selection. The relative retrotransposition frequency of pCEP4-L1RPneo in the absence of siRNA was set at 100%. The negative control pCEP4-L1.3neo-
JM111, an RC-L1 clone with two missense mutations in ORF1 rendering it inactive, showed no retrotransposition activity. (A) Results of the retrotransposition
achieved in HCT116 cells. The wild-type pCEP4-L1RPneo (or the negative control pCEP4-L1.3neo-JM111) was transfected alone or with the indicated amount of
siRNA. G418 colonies were fixed and stained 14 days post-selection with 4% Giemsa for visualization.
Nucleic Acids Research, 2005, Vol. 33, No. 3 853
human U1 snRNA promoter showed no decrease in luciferase
activity in the presence of 50-UTR siRNA.
While the in vitro ‘diced’ siRNA represents a pool of many
21–23 nt siRNAs spanning the 910 nt 50-UTR region, and thus
it is not possible to know which fraction of the pool can
actually function to target the homologous regions of the tran-
script for RNAi-mediated degradation, we have demonstrated
that the RNAi effect occurs only with transcripts that include
the 50-UTR and not the luciferase coding region itself.
In addition, in vitro ‘diced’ irrelevant siRNA from the
b-galactosidase coding region did not reduce the expression
of the hybrid transcript, providing further evidence that the
significant reduction in FF luciferase activity observed with
the pool of 50-UTR assay is due to sequence-specific targeting
of the hybrid transcript. Furthermore, the reduced luciferase
mRNA levels detected by semi-quantitative RT–PCR provide
additional evidence that the decreased luciferase activity is
due to reduction in the hybrid transcript, and not due to altered
translation of the hybrid reporter gene. Thus, our data is
in agreement with two other published reports that in vitro
cleavage of long dsRNA by recombinant DICER is a viable
method to produce sequence-specific siRNAs for gene silen-
cing experiments (46,47). Interestingly, the use of a single
siRNA (50-UTR #749) provided a more potent silencing effect
than a similar concentration of diced 50-UTR siRNA. Further-
more, the silencing effect of the synthetic 50-UTR siRNA was
very pronounced even at low concentrations, but we did not
observe the same silencing effect with diced siRNA at these
lower concentrations. One speculation for the increased
potency of the synthetic siRNA is that the functional 21mers
in the diced siRNA pool compete with the many non-
functional 21mers for target binding and active RISC forma-
tion. Indeed, we have observed a similar phenomenon when
targeting endogenous genes; namely that two weakly func-
tional siRNAs do not perform together as well as a single
efficient siRNA at lower concentrations (H. S. Soifer, unpub-
Although we demonstrated that siRNA homologous to the
50-UTR could target a hybrid reporter transcript for RNAi-
mediated degradation, it was not readily apparent that the
full length L1 transcript would be susceptible to RNAi.
This concern grew from the fact that the L1 RNA exists in
the cytoplasm as RNPs with multimerized p40 protein
encoded by its own ORF1 (3,4). Although the function of
the human p40 has not been assigned, one possibility is
that the RNPs protect the L1 from degradation by cytoplasmic
RNAses. Despite the co-localization of L1 RNA to RNPs,
however, siRNA targeting the 50-UTR produced >80% reduc-
tion in L1 retrotransposition activity in HCT116 cells at con-
centrations of ?10 nM. We observed a greater reduction of
retrotransposition activity in HeLa cells with the same con-
centrations of diced 50-UTR siRNA relative to the no siRNA
control. The increased response of HeLa cells to 50-UTR
siRNA might result from the hyper-triploid karyotype of
HeLa cells, thus providing more RNAi components compared
with HCT116 cells, which possess a near diploid karyotype
(48,49). Since we have little understanding for the role of
human p40 in the retrotransposition process, we can only
speculate that the siRNA is working at a stage before the
RNP assembly with L1 RNA. If the siRNA is capable of
targeting the L1 RNA after p40 multimerization and RNP
assembly, this would require association of the large RNP
with RISC producing a very large complex approaching
800 kDa. While p40 does contain a leucine zipper that
could mediate interactions with other proteins, there is no
evidence to suggest that the human L1 p40 interacts with
the RISC machinery.
In addition to targeting the 50-UTR, we also produced
in vitro ‘diced’ siRNA from the ORF1 region of the RC-L1
clone L1RP. The level of inhibition achieved with 10 nM of
diced ORF1 siRNA was significantly greater than the inhibi-
tion observed with a similar concentration of 50-UTR siRNA.
A numberof possibilities exist to explain the more pronounced
decrease in L1 retrotransposition in response to ORF1 siRNA.
The most obvious explanation is that the ORF1 siRNA pool
contains more functional siRNA molecules than the pool of
50-UTR siRNA. While there is some indication as to the ther-
duplex from a non-functional siRNA, such as 50-end duplex
stability, GC content, and internal Tm, we are still unable to
predict which set of 21mers from a given long dsRNA will be
effective siRNAs (50,51). Additionally, we cannot identify
the population of ‘diced’ siRNA based on the sequence of
the starting long dsRNA. Although biochemical evidence sug-
gests that DICER recognizes the stem loop structure of both
shRNA and micro RNA precursors and measures from the end
of the stem to cleave a 21–23 nt duplex, the manner in which
DICER cleaves long dsRNA ex vivo has not been established
(52) (H. S. Soifer, unpublished data). Another possibility is
that the stable secondary structure of the 50-UTR resulting
from higher GC content (58% 50-UTR versus 42% ORF1)
poses a barrier to RNAi. Indeed, strong localized secondary
structure surrounding the target site of the mRNA is known to
negatively affect RNAi (53).
In summary, we have presented evidence that the L1 retro-
transposon is susceptible to RNA interference. While our
study does not provide direct proof that RNAi functions to
control the activity of the remaining RC-L1s, we have pro-
vided proof of principle that the RNAi machinery can act on
an active RC-L1 and limit its retrotransposition activity using
a cell culture retrotransposition assay. Direct evidence that
RNAi limits the activity of L1s awaits the cloning of endo-
genous LINE-1 siRNAs from human cells. Efforts to clone the
small RNA population from cultured human cells failed to
detect L1 siRNA, suggesting that if endogenous L1 siRNAs
are produced, they may be present only during specific devel-
opmental stages (54). One requirement for the production of
L1 siRNA would be transcription of antisense L1 RNA that
could hybridize with L1 sense RNA to form either dsRNA or
siRNA. In fact, there is experimental evidence demonstrating
that large quantities of both sense and antisense L1 RNA of
variable size are present in the total RNA of a human terato-
more than 500 000 L1 sequences, it is possible that a subset
is transcribed by a nearby promoter into ds- or siRNA. This
arrangement, however, would still require the existence of two
opposing promoters to transcribe both sense and antisense L1
sequence. Alternatively, the production of sense/antisense L1
dsRNA might take advantage of a unique feature of the
L1 50-UTR, namely the existence of an internal promoter
that transcribes L1 sense RNA and an antisense promoter
within nucleotides +400 to +600 of the 50-UTR that transcribes
854 Nucleic Acids Research, 2005, Vol. 33, No. 3
L1 sequence in the opposite direction. In cell lines where the
50-UTR sense promoter shows transcriptional activity, the L1
ASP is also transcriptionally active, albeit at lower levels (43).
A recent report demonstrates that transcription from the L1
ASP is dependent on the transcription factor RUNX3 (44). In
addition to characterizing endogenous L1-derived siRNAs
that may arise from the L1s 50-UTR, the establishment of
conditional RNAi knock-out mice and derived fibroblast
lines will permit more extensive analysis of the retrotranspo-
son activity than can be achieved with the current DICER and
AGO2 null mouse models (31,56).
We would like to thank Dr John V. Moran (University
of Michigan) and Dr Haig H. Kazazian (University of
Pennsylvania) for generously providing the pCEP4-based
retrotransposon plasmids used in this study. We also thank
members of the Rossi Lab for helpful comments. This work
was supported by a post-doctoral fellowship awarded to H.S.S.
NLB HL074704 to J.J.R. Funding to pay the Open Access
publication charges for this article was provided by grant
NLB HL074704 to J.J.R.
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