Uncoupling of RNAi from active translation
in mammalian cells
SHUO GU AND JOHN J. ROSSI
Division of Molecular Biology, Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope,
Duarte, California 91010
Small inhibitory RNAs (siRNAs) are produced from longer RNA duplexes by the RNAse III family member Dicer. The siRNAs
function as sequence-specific guides for RNA cleavage or translational inhibition. The precise mechanism by which siRNAs
direct the RNA-induced silencing complex (RISC) to find the complementary target mRNA remains a mystery. Some biochemical
evidence connects RNAi with translation making attractive the hypothesis that RISC is coupled with the translational apparatus
for scanning mRNAs. Such coupling would facilitate rapid alignment of the siRNA antisense with the complementary target
sequence. To test this hypothesis we took advantage of a well-characterized translational switch afforded by the ferritin IRE-IRP
to analyze RNAi mediated cleavage of a target mRNA in the presence and absence of translation. Our results demonstrate that
neither active translation nor unidirectional scanning is required for siRNA mediated target degradation. Our findings demon-
strate that nontranslated mRNAs are highly susceptible to RNAi, and blocking scanning from both the 5? and 3? ends of an mRNA
does not impede RNAi. Interestingly, RNAi is about threefold more active in the absence of translation.
Keywords: RNA interference (RNAi); small interfering RNA (siRNA); translation; iron responsive element (IRE)
RNA interference (RNAi) is a phenomenon in which
double-stranded RNA triggers the silencing of target gene
expression by inducing sequence-specific target mRNA deg-
radation (Fire et al. 1998). An important intermediate in the
RNAi pathway are small interfering RNAs (siRNAs) which
are duplexes of 21–23 nucleotides containing two unpaired
nucleotides at the 3?-end of each strand (Hamilton and
Baulcombe 1999; Zamore et al. 2000). Strong inhibition of
target gene expression using synthetic or expressed siRNAs
has been demonstrated in mammalian cells by many groups
(Elbashir et al. 2001; McManus and Sharp 2002; Scherer
and Rossi 2003).
A critical step in RNAi function is target site recognition,
in which the antisense strand derived from the siRNA serves
as a guide for RISC to find the complementary target se-
quence (Hammond et al. 2000; Martinez et al. 2002). The
precise mechanism by which this process occurs remains to
be determined. There are at least two general models: ran-
dom access and directed scanning. Very small amounts of
siRNA can efficiently induce targeted mRNA degradation
(Dykxhoorn et al. 2003), therefore it is highly likely that
RISC functions catalytically.
In considering mechanisms by which RISC targets
mRNAs, a unidirectional or bidirectional scanning process
could facilitate more rapid alignment of the siRNA anti-
sense with the complementary target sequence. If a scan-
ning model is correct, it is likely that RISC function would
be coupled with some cellular process that effectively scans
mRNAs, such as translation (Merrick 1992; Sachs et al.
1997). Some biochemical evidence already exists which con-
nects RNAi with translation in that RISC components have
been found to co-purify with translation factors such as
ribosomal proteins L5, L11, as well as 5S rRNA (Hammond
et al. 2001; Caudy et al. 2002; Ishizuka et al. 2002; Caudy et
al. 2003; Pham et al. 2004). Furthermore, siRNAs were
found associated with polyribosomes in the protozoan Try-
panosome brucei (Djikeng et al. 2003). We therefore asked
whether or not the mammalian siRNA pathway of RNAi is
linked with translation. As a model system for addressing
this question, we have taken advantage of the factors in-
volved in the regulation of ferritin translation (Theil 1990;
Hentze and Kuhn 1996). When the intracellular iron con-
centration is low, the iron regulatory protein 1 (IRP-1)
Reprint requests to: John J. Rossi, Division of Molecular Biology,
Graduate School of Biological Sciences, Beckman Research Institute of the
City of Hope, 1450 E. Duarte Rd., Duarte, CA 91010; e-mail: jrossi@coh.
email@example.com; fax: (626) 301-8271.
Article published online ahead of print. Article and publication date are
RNA (2005), 11:38–44. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 RNA Society.
binds to an iron-responsive element (IRE) located in close
proximity to the cap structure of the ferritin mRNAs. This
IRP1-IRE complex represses translation by blocking the re-
cruitment of the small ribosomal subunit to these mRNAs
(Muckenthaler et al. 1998). To take advantage of this rela-
tively simple system, we have created a reporter gene con-
struct under the regulation of IRE/IRP-1 system. This IRE-
IRP-1 system can be utilized as a molecular switch to regu-
late translation, thereby allowing the testing of siRNA
function in the absence of active translation. An additional
feature of this system is that it allows the manipulation of a
specific reporter gene construct without impacting on glo-
bal translation. We demonstrate here that neither active
translation nor unidirectional scanning is required for
siRNA-mediated target degradation. The implications of
our findings are that nontranslated mRNAs are susceptible
to RNAi, suggesting that RISC identification of target RNAs
is not actively linked with mRNA translation.
In an effort to regulate translation of a reporter gene con-
struct, we designed a vector using the Pol II U1 promoter to
drive expression of an EGFP gene in which the iron re-
sponse element (IRE) was inserted upstream of the EGFP
coding sequence. This construct is designated as pU1-IRE-
GFP. A similar construct, pU1-GFP, lacking the IRE was
constructed as a control (Fig. 1A,B). Notably, the IRE is
inserted 28 base pairs (bp) downstream of the cap site.
Close proximity to the cap structure has been shown to be
important for the IRE/IRP-1 system to function as a trans-
lational regulator (Koloteva et al. 1997). Translational regu-
lation of the pU1-IRE-GFP was achieved by the addition of
Hemin (as a source of iron) or deferoxaminemesylate salt
(as an iron chelator) following transfection of the reporter
plasmid into HEK293 cells. EGFP expression from pU1-
IRE-GFP was greatly reduced when the iron concentration
was reduced with deferoxaminemesylate salt (Figs. 1C and
2B,C). RT-PCR analyses of the IRE-GFP mRNA revealed
that the mRNA level remains unchanged from conditions in
which iron is present (Fig. 1D), demonstrating the repres-
sion of EGFP expression is solely due to a block in trans-
lation. EGFP expression from the pU1-GFP control vector
was not affected by the presence or absence of iron (Fig.
1C,D). Consistent with previous reports, our data con-
firmed that the IRE-IRP1 system functions as a translational
switch (Hentze et al. 1987).
To address the question of whether or not RNAi function
is coupled with translation, a synthetic siRNA targeting the
EGFP coding region was tested for RNAi in the pU1-IRE-
FIGURE 1. Iron response element (IRE) and IRP regulate translation in HEK293 cells. (A) sequence and computer-modeled secondary structure
of the human ferritin H-chain IRE and synthetic oligonucleotides used in this study. The sequence recognized by the iron regulatory protein (IRP),
is highlighted in red. (B) Designs of the two constructs used in this study. (C) Three-hour post-transfection of the constructs in HEK293 cells,
Hemin (50 µM final concentration), or deferoxaminemesylate salts (100 µM final concentration) were added to the medium and uptake was
allowed to proceed for 21 h. EGFP expression 24 h after transfection is depicted. (D) EGFP mRNA levels analyzed by RT-PCR. GAPDH, served
as internal control, and the RT-PCR products are also depicted. U1-GFP plasmid DNA and total RNA from nontransfected HEK293 cells were
used as standards and positive controls for EGFP and GAPDH, respectively. To control for DNA contamination, minus RT PCR reactions were
carried out for each RNA preparation.
Active translation is not required for RNAi
GFP and pU1-GFP cells in the presence and absence of iron.
In co-transfections of the siRNA with the expression vectors
this siRNA effectively reduced EGFP expression from both
constructs under both high and low iron concentrations
(Fig. 2). These results demonstrate that siRNA mediated
RNAi is fully functional when translation of the target
mRNA is active or severely blocked by greater than 80%
(Fig. 2B). Interestingly, quantitative real-time RT-PCR
analyses of the EGFP mRNA revealed that the siRNA in-
duced about a fourfold reduction of the actively translated
IRE-GFP mRNA whereas an approximate 13-fold reduction
of this mRNA was achieved when translation was blocked.
These results suggest that in the absence of translation the
mRNA may be more accessible to RNAi. This increase in
siRNA efficacy during translational inhibition was consis-
tently observed in three independent experiments (Fig. 2).
Moreover, the mRNA levels analyzed in these studies were
normalized with GAPDH mRNA levels, which were un-
changed; demonstrating specificity in the siRNA-mediated
targeting of EGFP.
There have been several reports suggesting that high con-
centrations of siRNA can result in off-target effects or ac-
tivate genes in the interferon pathway (Sledz et al. 2003;
Scacheri et al. 2004). To validate the specificity of our re-
sults, we tested another synthetic siRNA targeting a differ-
ent sequence in EGFP. For these studies we utilized a syn-
thetic siRNA that we have independently demonstrated to
be a potent trigger for RNAi-mediated destruction of the
EGFP mRNA (D. Kim, M. Behlke, S. Rose, M. Chang, S.
Choi, and J.J. Rossi, unpubl.). With this siRNA we utilized
a final concentration of only 2 nM and repeated the trans-
fection experiments described above. The results obtained
FIGURE 2. siRNA mediated RNAi is fully functional when translation of the target mRNA is active or blocked. (A) EGFP expression from
U1_GFP and U1_IRE_GFP under various conditions. Hemin (iron source) labeled as H or deferoxaminemesylate salts (iron chelator) labeled as
D and siRNA targeting the EGFP coding region were added to the culture medium as indicated. (B) Cells were lysed 24 h after treatment, and
levels of EGFP and an internal control ?-actin were obtained by sequential immunoblotting using anti-eGFP and anti-?-actin antibodies. Lanes
(from left to right): U1_GFG plus Hemin; U1_GFP plus deferoxaminemesylate salts; U1_GFP treated with siRNA; U1_GFP treated with both
siRNA and deferoxaminemesylate salts; U1_IRE_GFP plus Hemin; U1_IRE_GFP plus deferoxaminemesylate salts; U1_IRE_GFP treated with
siRNA; U1_IRE_GFP treated with both siRNA and deferoxaminemesylate salts. (C) Summary of EGFP expression and mRNA levels for each
sample. The mean levels of EGFP expression were measured by Dot density assays of the immunoblots (B) after normalization with the internal
control ?-actin. The EGFP mRNA levels were determined using real time RT-PCR after normalization with GAPDH mRNA levels. EGFP
expression and mRNA levels from the U1-GFP plus Hemin in the absence of siRNA were assigned a value of 100 percent. Quantitations of EGFP
values and mRNA relative to the U1-GFP values are plotted. The results are derived from averaging values from triplicate experiments. Values
for EGFP expression for all of the samples are (from left to right): 100 ± 0%, 93.9 ± 6.6%, 27.3 ± 6.1%, 22.8 ± 5.1%, 108.1 ± 18.8%, 17.7 ± 4.4%,
27.5 ± 8.3%, 3.1 ± 2.1%. Values for mRNA level of all samples are (from left to right): 100 ± 0%, 107.4 ± 1.7%, 18.5 ± 6.4%, 16.6 ± 5.3%,
110.0 ± 19.2%, 107.3 ± 15.3%, 28.9 ± 6.2%, 8.1 ± 2.6%.
Gu and Rossi
RNA, Vol. 11, No. 1
with this more potent siRNA trigger were similar to those
obtained with the first siRNA although the second siRNA
worked at a much lower concentration (data not presented).
To further substantiate the conclusion that siRNA trig-
gered RNAi is translation independent, we blocked trans-
lation via a different approach, using the translational elon-
gation inhibitor Hygromycin B (Gonzalez et al. 1978). As
we observed using the IRE-IRP1 approach, a significant
reduction of target mRNA was achieved by siRNA treat-
ment in Hygromycin B treated cells (Fig. 3).
Although translation is not required for siRNA, it is pos-
sible that RISC may scan messages 3? to 5?. To test this
possibility we designed a construct harboring the IRE in
both the 5?-UTR and 3?-UTRs of the EGFP mRNA. Under
conditions of low iron the IRP-1 should bind to both ele-
ments that in turn would block a unidirectional scan from
either end (Fig. 4A). Utilizing this double IRE containing
construct we observed efficient RNAi under both condi-
tions of high and low iron (Fig. 4B,C). These data suggest
that unidirectional scanning of mRNAs, initiating either at
the 5? or 3? ends of the transcripts, is not the mechanism by
which RISC functions.
There are several cellular mechanisms in which an RNA
guide sequence is used to identify the target RNA sequence.
These include translation (Sachs et al. 1997), pre-mRNA
splicing (Nilsen 1994), RNA editing (Smith et al. 1997),
telomere synthesis (Cech 2004) and snoRNA mediated pre-
rRNA site-specific modifications (Lafontaine and Tollervey
1998). For each of these processes, a complex of proteins
associates with the guide RNA that provides the specificity
for the process. In the case of RNAi, the antisense strand
selected from the siRNA duplex guides RISC to the target
sequence. Based upon the reported potencies of RNAi,
identification of the target is an efficient process, which
presumably would make it a diffusion independent process.
Since message-specific degradation by RNAi has been pur-
ported to be a cytoplasmic process (Zeng and Cullen 2002),
we devised a set of experiments to ask whether or not RISC
is associated with the translational apparatus, thereby pro-
viding a scaffold for scanning mRNAs from the 5? to 3? end.
Several relevant but conflicting sets of data have been pub-
lished in the past few years. Zamore and coworkers previ-
ously showed that RNAi activity is not attenuated when
translation is blocked by several antibiotics in Drosophila
lysates, indicating translation is uncoupled from RNAi ac-
tivity in vitro in Drosophila (Zamore et al. 2000). In con-
trast, there are observations that untranslated mRNAs are
recalcitrant to RNAi in Drosophila oocytes in vivo (Ken-
nerdell et al. 2002). The insensitivity to RNAi of maternal,
untranslated mRNAs in oocytes has been attributed to pro-
tective mechanisms that sequester these mRNAs from both
translation, and hence RNAi (Kennerdell et al. 2002). Other
studies utilizing RNAi to target viruses with RNA genomes
are also contradictory. Hu and colleagues provided evidence
that HIV-1 RNA genomes encapsulated in viral particles
during the early stages of viral entry and reverse transcrip-
tion are relatively insensitive to RNAi (Hu et al. 2002)
whereas Jacque and coworkers observed that genomic HIV
RNA within a nucleoprotein complex is still susceptible to
RNAi (Jacque et al. 2002). In the present study we took
advantage of a well-characterized translational switch af-
forded by the ferritin IRE-IRP1 and compared RNAi results
from this system with antibiotic mediated inhibition of
translation. Our results demonstrate that an untranslated
target is as susceptible, if not more susceptible to RNAi than
a translated target, indicating that in live cells active trans-
lation is not required for siRNA function. Interestingly, in-
hibition of translation initiation rendered by the IRE-IRP1
complex would leave fewer ribosomes on the target mRNA.
In contrast, Hygromycin mediated arrest of translational
elongation would presumably leave more ribosomes along
the target mRNA (Todorov et al. 1977). RNAi function was
not impaired in either case, demonstrating the recruitment
of RISC to the target mRNA does not
require active translation.
In addition to investigating the link
between translation and RNAi, we took
advantage of the ferritin IRE-IRP-1 sys-
tem to test whether or not unidirec-
tional 5? to 3? or 3? to 5? scanning was
required for RNAi. Collectively the re-
sults we have obtained suggest that
RNAi can take place in the absence of
active translation apparatus and does
not involve a unidirectional scanning
process. Our results are not in total con-
tradiction with the biochemical evi-
dence that links RISC to components of
the translational apparatus, but they
clearly demonstrate that active transla-
FIGURE 3. Translational elongation mediated by Hygromycin does not protect transcripts
from RNAi mediated degradation. (A) Three hours after U1_GFP was transfected into HEK293
cells, Hygromycin (10 nM final concentration) was added to the medium with or without
siRNA against EGFP. EGFP expression was monitored 21 h later. (B) EGFP mRNA levels were
analyzed by RT-PCR as described in the legend to Figure 2.
Active translation is not required for RNAi
tion is not required for RNAi. The actual mechanism by
which RISC efficiently finds the target sequence is still un-
known. An attractive possibility is that RNAi may be linked
to another RNA degradation pathway, the recently de-
scribed RNA processing bodies, or P bodies (Sheth and
Parker 2003). These could serve as sites for co-localizing
mRNAs with the degradation components. The problem
of how mRNAs get to P bodies is currently unknown. RNAi
functions to reduce target RNA levels by impacting on the
steady state levels of the target mRNA. Thus, RNAs may
be cycled through P-bodies following a round of transla-
tion or cycled to P-bodies when translation is blocked, pro-
viding a possible explanation for our results. The obser-
vation that siRNA efficacy is increased during translational
inhibition implies that RNAi and translation are not totally
independent processes. One rational hypothesis is that
one or more cellular factors are shared by the translation
apparatus and RISC, thereby establishing a competitive re-
lationship between those two pathways. Finally, the obser-
vation that micro RNAs which normally direct translational
inhibition can be converted to siRNAs that direct cleavage
by simply changing the base-pairing properties with the
target (Doench et al. 2003) suggests that these two path-
ways share a common mechanism for target identifica-
tion. It would seem that in the case of micro RNAs, the
block to translation may also occur prior to ribosome
scanning of the mRNA. Further understanding of the in-
tracellular localization of RISC complexes and their rela-
tion to the translational machinery is of paramount impor-
MATERIALS AND METHODS
DNA constructs and siRNAs
Both strands of IRE were chemically synthesized (sense strand:
antisense strand: CCCCAAGCTTGTCCAAGCACTGTTGAAGCA
AAGCTTGGGG), annealed, purified, and restriction digested by
HindIII. The products were inserted into a HindIII site 28 bp
downstream of transcription start site of the human U1 snRNA
promoter, which was cloned upstream of an EGFP-coding se-
quence in a pBluescript vector. A similar approach was used to
generate the double IRE construct by insertion of another IRE-like
sequence within a unique XhoI site downstream of the EGFP stop
codon. The siRNAs were synthesized by the City of Hope DNA/
RNA synthesis facility. The sequence of the sense strand of siRNA-
GFP is 5?-GCUGACCCUGAAGUUCAUCdTdT; the antisense
strand sequence of siRNA-GFP is 5?-GAUGAACUUCAGGGUCA
Hemin (Cat No. 51280) and Deferoxaminemesylate (Cat No.
D9533) were purchased from SIGMA-ALDRICH, Inc.
Cell culture and transfections
Adherent HEK-293 cells were grown in 10% FBS in DMEM,
supplemented with glutamine in the presence of antibiotics. All
transfection assays were done using Lipofectamine 2000 (Invitro-
gen) following the manufacturer’s protocol. HEK-293 cells at
ninety percent confluency were transfected in six-well plates with
0.5 µg reporter gene construct and 50 nM siRNA (final concen-
tration) unless specified otherwise. Carrier DNA was used to bring
the total amount of nucleic acid transfected to each well to 4 µg.
FIGURE 4. RNAs containing IRE duplications at the 5? and 3? end are still susceptible to siRNA triggered RNAi. (A) Schematic representation
of the Double IRE construct and how it would be protected when the iron concentration is low. (B) Three hours after the U1_Double_IRE_GFP
was transfected into HEK293 cells, Hemin (iron source labeled as H) or deferoxaminemesylate salts (iron chelator labeled as D) were added to
the medium with or without siRNA against EGFP. EGFP expression was monitored 24 h post-transfection. (C) EGFP mRNA levels are analyzed
by RT-PCR as described in the legend to Figure 2.
Gu and Rossi
RNA, Vol. 11, No. 1
RNA extraction and real-time RT-PCR
Total RNA was isolated using RNA STAT-60 (TEL-TEST B, Inc.)
following the product protocol. The DNA-free kit (Ambion Cat #
1906) was used to purify total RNA from contaminating DNA.
Oligo-dT was used to produce the cDNAs using a commercial
Reverse Transcriptase kit (Invitrogen) following manufacturer’s
protocol. cDNA from each sample was used as template for the
subsequent PCR and real-time PCR analyses. Real-time PCR re-
actions were performed in a Bio-Rad Light Cycler using Cyber
Green to monitor amplification. The PCR primers are: 5?-ACG
TAAACGGCCACAAGTTC (sense) and 5?-AAGTCGTGCTGCTT
CATGTG (antisense). GAPDH was used as internal control, and
was amplified using the following primers: 5?-CATTGACCTCA
ACTACATG (sense) and 5?-TCTCCATGGTGGTGAAGAC (anti-
Serial dilutions of the U1-GFP plasmid were used to produce
the standard curve and to determine the copy number of GFP
transcripts in the samples. Serial dilutions of total RNA from
nontransfected HEK293 cells were used to produce the standard
curve for GAPDH. In all of the assays, linear correlation coeffi-
cients (r) for the standard curve were greater than 0.97.
SDS-PAGE and Western blotting
Cell extract samples were denatured in loading buffer (final con-
centration: 50 mM Tris, 5% SDS, 10% glycerol, 2% 2-mercapto-
ethanol, 0.01% Bromophenol blue dye) for 5 min at 95°C and
separated in 10% SDS-PAGE gels. The denatured proteins were
then electro-transferred onto a PVDF membrane which was
blocked with 5% fat-free milk powder in PBS and 0.5% Tween 20
for 1h. An anti-EGFP antibody (diluted 1:4000, Clonetech), and
an anti-?-actin antibody (diluted 1:2500, Sigma) were used se-
quentially. Following three washes of 5 min in PBS, a secondary
antibody (HRP-anti-mouseIgG, Sigma, dilution 1:10000) was in-
cubated with the blots for 1 h at room temperature, followed by
three 5-min washes in PBS. Antibody-bound proteins were visu-
alized using the ECL Western blotting analysis system (Amersham,
This work was supported by NIH grants to JJR: AI29329, AI42552,
and HL074704. The authors wish to thank Dongho Kim for pro-
viding the EGFP siRNAs and members of the Rossi Lab for critical
discussions of this work.
Received August 18, 2004; accepted October 8, 2004.
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