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ArticleVirus Sensor RIG-I Represses RNA Interferenceby Interacting with TRBP through LGP2
in Mammalian Cells
Tomoko Takahashi 1 , Yuko Nakano 1 , Koji Onomoto 2 , Mitsutoshi Yoneyama 2
and Kumiko Ui-Tei 1,3, *
1
2
3

*

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033,
Japan; ttakahas@bs.s.u-tokyo.ac.jp (T.T.); yuko.f.nakano@gmail.com (Y.N.)
Division of Molecular Immunology, Medical Mycology Research Center, Chiba University,
Chiba 260-8673, Japan; konomoto@chiba-u.jp (K.O.); myoneyam@faculty.chiba-u.jp (M.Y.)
Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences,
The University of Tokyo, Chiba 277-8561, Japan
Correspondence: ktei@bs.s.u-tokyo.ac.jp; Tel.: +81-3-5841-3043

Received: 22 September 2018; Accepted: 17 October 2018; Published: 19 October 2018


Abstract: Exogenous double-stranded RNAs (dsRNAs) similar to viral RNAs induce antiviral
RNA silencing or RNA interference (RNAi) in plants or invertebrates, whereas interferon (IFN)
response is induced through activation of virus sensor proteins including Toll like receptor 3(TLR3) or retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) in mammalian cells. BothRNA silencing and IFN response are triggered by dsRNAs. However, the relationship between
these two pathways has remained unclear. Laboratory of genetics and physiology 2 (LGP2) is oneof the RLRs, but its function has remained unclear. Recently, we reported that LGP2 regulates
endogenous microRNA-mediated RNA silencing by interacting with an RNA silencing enhancer,
TAR-RNA binding protein (TRBP). Here, we investigated the contribution of other RLRs, RIG-I andmelanoma-differentiation-associated gene 5 (MDA5), in the regulation of RNA silencing. We foundthat RIG-I, but not MDA5, also represses short hairpin RNA (shRNA)-induced RNAi by type-I IFN.
Our finding suggests that RIG-I, but not MDA5, interacts with TRBP indirectly through LGP2 tofunction as an RNAi modulator in mammalian cells.
Keywords: RNA interference; RLRs; RIG-I; LGP2; TRBP; virus sensor; dsRNA

1. Introduction
During virus infection, double-stranded RNAs (dsRNAs), which are generated as viral replicationintermediates, are recognized by virus sensor proteins [1,2]. One of the Toll like receptor family proteins(TLRs), TLR3 [3], and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) function as virus RNA
sensor proteins in endosome and cytoplasm of mammalian cells, respectively [4,5]. RLRs include threeproteins, RIG-I, melanoma-differentiation-associated gene 5 (MDA5), and laboratory of genetics andphysiology 2 (LGP2). RIG-I and MDA5 are activated by different types of viral RNAs; RIG-I is activatedby 50 -triphosphate- or 50 -diphosphate-containing RNA or small RNA duplexes [6–10]; and MDA5 is
activated by long dsRNA duplexes [11]. Activated virus sensors transfer the signal to downstream
molecules, leading to the production of type-I interferon (IFN). Secreted IFN induces hundredsof IFN-stimulated genes including TLR3 and RLRs to establish potent antiviral states of the cells.
Both RIG-I and MDA5 have caspase recruitment domains (CARDs), which are necessary for signaltransfer to downstream molecules [1,2]. However, LGP2 does not have CARD, thus the role of
LGP2 is controversial. Previous reports show positive or negative effects of LGP2 on RLR signaling.
Genes 2018, 9, 511; doi:10.3390/genes9100511

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LGP2 positively regulates RIG-I- and MDA5-mediated antiviral responses [12], whereas LGP2
negatively regulates RIG-I-mediated dsRNA recognition [13–15]. Furthermore, type I IFN production
is promoted in Lgp2−/− mice in response to polyI:C stimulation and vesicular stomatitis virus (VSV)
infection, whereas its response to encephalomyocarditis virus (EMCV) is suppressed [16].
RNA silencing or RNA interference (RNAi) is a posttranscriptional gene silencing mechanism
conserved in wide variety of organisms, directed by microRNAs (miRNAs), a class of approximately
22-nucleotide (nt)-long non-coding RNAs. Human genome encodes approximately 2000 miRNAs,
which regulate gene expression patterns of multiple genes [17]. Many reports demonstrate that
mutation or deletion of one miRNA induces serious diseases, indicating the importance of gene
regulation by miRNAs [18–20]. Primary miRNA transcripts (pri-miRNAs) are cleaved into miRNA
precursors (pre-miRNAs) by an endoribonuclease, Drosha, in the nucleus. The pre-miRNAs
are exported into the cytoplasm by exportin-5 [21,22], and further processed by Dicer, anotherendoribonuclease, to yield dsRNA with 2-nt 30 -overhangs [23]. The trans-activation-responsive
region (TAR)-RNA binding protein (TRBP) is an RNA silencing enhancer, and functions to recruit
pre-miRNA into Dicer [24,25]. TRBP was originally identified as a protein that interacts with the humanimmunodeficiency virus (HIV)-1 TAR RNA [26] and was later demonstrated to regulate IFN-induced
protein kinase R (PKR), a kinase involved in the cellular response to viral infection [27,28]. Smallinterfering RNA (siRNA) is another type of small non-coding RNA derived from long dsRNA thatfunctions in a similar manner to miRNA. Long dsRNA induces RNAi or RNA silencing in plants andinvertebrates [29,30], but activates IFN response in mammals [1,2]. Small RNA duplexes are loaded
onto the Argonaute (AGO) protein in the RNA-induced silencing complex (RISC), and unwound into
single-stranded RNAs. A guide strand of miRNA/siRNA resides in the RISC represses target mRNAs
with sequence complementarities [23,31,32].
Recently, LGP2 has been shown to inhibit Dicer-dependent processing of long dsRNA and block
mammalian RNAi [33], and we and others reported that TRBP interacted with LGP2 but not with
RIG-I or MDA5 [34,35]. Furthermore, we revealed that LGP2 interacts with TRBP, by competing forits dsRNA-binding sites [34]. This competitive binding represses the recruitment of TRBP-boundpre-miRNAs into Dicer, resulting in the decrease of mature miRNA levels and subsequent RNA
silencing activities. Thus, the endogenous gene expression might be regulated by miRNAs that are
well regulated by LGP2–TRBP interaction during viral infection. Komuro et al. further revealed
that TRBP increases Cardiovirus-triggered interferon promoter activity only when LGP2 and MDA5
are co-expressed but not MDA5 alone [35]. Thus, TRBP is important for Cardiovirus-triggered IFN
responses mediated by the LGP2–MDA5 interaction, but is not involved in Sendai virus-triggered
IFN response mediated by RIG-I. Based on these results, we examined the contribution of RIG-I andMDA5 for the regulation of RNA silencing. Our results revealed that RIG-I, but not MDA5, regulates
short-hairpin RNA (shRNA)-induced RNAi when the expression levels were increased by type-I IFN
treatment. The immunoprecipitation assays revealed that both RIG-I and MDA5 did not interact
with TRBP directly. However, RIG-I, but not MDA5, interacted with TRBP indirectly through LGP2.
Thus, our finding suggests that RIG-I may function as an RNAi modulator in collaboration with LGP2
in mammalian cells.
2. Materials and Methods
2.1. Cell Culture
Human HeLa wild-type, HeLa RIG-I−/− , HeLa LGP2−/− , HeLa TRBP−/− , and HEK293T cells
were cultured in Dulbecco’s Modified Eagle’s Medium (Wako, Osaka, Japan) containing 10% fetal
bovine serum (Gibco, Waltham, MA, USA) at 37 ◦ C with 5% CO2 .

Genes 2018, 9, 511

3 of 132.2. Oligonucleotides
siRNA oligonucleotides used for knockdown of AGO2, RIG-I, MDA5, or LGP2 were chemicallysynthesized as dsRNAs (Shanghai GenePharma, Shanghai, China). Guide and passenger strand RNA
oligonucleotides for electrophoretic mobility shift assay (EMSA) were chemically synthesized (Proligo,
St. Louis, MO, USA), and purified by polyacrylamide gel electrophoresis. The guide strand was labeledwith [γ-32 P] ATP (6000 Ci/mmol; Perkin Elmer, Waltham, MA, USA). Annealing was performed byincubation at 95 ◦ C for 5 min, and gradually decreasing the temperature to 25 ◦ C. The annealing was
confirmed using 15% polyacrylamide gel electrophoresis in Tris–borate–EDTA (TBE) buffer, which candiscriminate 21-base-pair double-stranded siRNA from 21-nt single-stranded RNA. Almost all RNAs
were recovered as dsRNAs. The RNA sequences are shown in Table S1.
2.3. Plasmid Construction
The expression plasmids of N-terminal FLAG- or C-terminal Myc-tagged TRBP (FLAG-TRBP,
or TRBP-Myc) were constructed as described previously [34,36,37]. The plasmids encoding N-terminal
FLAG-tagged RIG-I or MDA5 were kindly provided from Dr. T. Fujita (Institute for Frontier Life
and Medical Sciences, Kyoto University) [38], and the C-terminal FLAG-tagged LGP2 (LGP2-FLAG)
was obtained from Addgene (plasmid #27234, Cambridge, MA, USA). The C-terminal Myc-tagged
RIG-I, or LGP2 (RIG-I-Myc, LGP2-Myc) was constructed from FLAG-tagged RIG-I or LGP2 expression
plasmids by PCR with primers containing Myc-tag sequence. The primer sequences are shown in
Table S2.
2.4. shRNA-Induced RNAi Activity Assay
Short hairpin RNA (shRNA)-induced RNAi activity was measured with a luciferase reporter
assay. A HeLa cell suspension (1.0 × 105 cells/mL) was inoculated in 24-well plates with or without
human type I IFN (IFN-α1; Cell Signaling, Danvers, MA, USA) 1 day before transfection. Cells weretransfected with 0.5 µg of pGL3-Control vector (Promega, Madison, WI, USA) encoding the firefly
luciferase gene, 0.1 µg of pRL-SV40 vector (Promega) encoding the Renilla luciferase gene, or 5 ng ofpSilencer-3.1-H1-puro vector encoding shRNA against the firefly luciferase with Lipofectamine 2000
reagent (Invitrogen, Carlsbad, CA, USA). In the RNAi activity assay, we used shRNA, since theDicer/TRBP complex is necessary for shRNA cleavage but not for siRNA. Specific siRNAs against
each gene for the knockdown (25 pmol) were transfected into the cells 1 day before transfectionof the pGL3-Control, pRL-SV40, and pSilencer-3.1-H1-puro vectors. Each siRNA sequence against
firefly luciferase, Ago2, RIG-I, MDA5, or LGP2 was satisfying the conditions of highly functional
siRNA sequences demonstrated previously, and designed by siDirect 2.0 [39,40]. siRNA against green
fluorescent protein (GFP) was used as control. The transfected cells were lysed with 1 × passive
lysis buffer (Promega) 24 h following transfection. Luciferase activity was measured using theDual-Luciferase Reporter Assay System (Promega, Madison, WI, USA), and the fold change of firefly
luciferase activity normalized to Renilla luciferase (firefly luciferase activity/Renilla luciferase activity)
was determined.
2.5. Western Blot
The samples were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membraneusing the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The membrane was blocked
for 1 h in Tris-buffered saline-Triton X-100 or Tween 20 (TBS-T; 20 mM Tris-HCl [pH 7.5], 150 mM NaCl,
0.2% Triton X-100, or 0.1% Tween) supplemented with 5% Difco skim milk (Becton, Dickinson andCompany, Franklin Lakes, NJ, USA), and incubated with specific antibodies in Can Get Signal
immunoreaction enhancer solution (TOYOBO, Osaka, Japan) at 4 ◦ C overnight. Anti-Myc or anti-FLAG(Cell Signaling, Danvers, MA, USA), anti-AGO2 (Wako, Osaka, Japan), and anti-α-tubulin antibodies(ICN/CAPPEL Biomedicals, Santa Ana, CA, USA) were used. Anti-human RIG-I, MDA5, and LGP2

Genes 2018, 9, 511

4 of 13antibodies were generated by immunizing rabbits with a synthetic peptide [41]. The membrane waswashed three times with TBS-T, and reacted with HRP-linked anti-rabbit or anti-mouse antibody(GE Healthcare, Chicago, IL, USA) at room temperature for 1 h. After being washed three times
with TBS-T, the membrane was incubated with ECL Prime Western Blotting Detection Reagent(GE Healthcare, Chicago, IL, USA) and visualized with the LAS3000 system (Fujifilm, Osaka, Japan).
2.6. Quantitative RT-PCR
Total RNA was extracted by RNeasy mini kit (QIAGEN, Hilden, Germany) and treated with
DNase I. A half µg of the total RNA was used for cDNA synthesis by Transcriptor High Fidelity cDNA
kit (Roche, Basel, Switzerland). Quantitative RT-PCR was performed by FastStart Universal SYBRGreen Master (Roche, Basel, Switzerland) by StepOnePlus realtime PCR system (Applied Biosystems,
Foster City, CA, USA). The sequences of the used primers are shown in Table S3.
2.7. Electrophoresis Mobility Shift Assay (EMSA)
Purification of the recombinant RIG-I, MDA5, and LGP2 proteins was performed as described
previously [8,14]. EMSAs were performed in binding buffer (20 mM Tris [pH 8.0], 1.5 mM MgCl2 ,50 mM NaCl, 1.5 mM DTT, 100 ng/µL sonicated salmon sperm DNA, 5% glycerol, and 0.4 U/mL
RNasin [Promega, Madison, WI, USA]). Each purified protein (3 µM) was incubated with 0.5 nM
siRNA containing 32 P-labeled guide strand for 30 min on ice. The samples were electrophoresed on a
9% polyacrylamide gel in 0.25 × TBE buffer. For quantification, gels were exposed to a Fuji imaging
plate and scanned with a Typhoon image analyzer (GE Healthcare, Chicago, IL, USA).
2.8. ImmunoprecipitationA HEK293T cell suspension (2 × 106 cells/dish) was plated into a 6-cm dish 1 day before
transfection, and cells were transfected with each combination of the plasmids. Cells were washedwith PBS and lysed in cold lysis buffer (50 mM, Tris-NaOH [pH 8.0], 150 mM NaCl, 1 mM Na3 VO4 ,1% NP-40, and complete protease inhibitor) 48 h following transfection. For immunoprecipitation,
the cell lysate was mixed with 30 µL of Protein G Dynabeads (Life Technologies, Carlsbad, CA, USA)and rotated at 4 ◦ C for 2 h with 2.5 µg of mouse anti-FLAG (Sigma, St. Louis, MO, USA) or
mouse anti-Myc antibody (Calbiochem, Burlington, MA, USA), or 2.5 µg of mouse IgG (Santa Cruz
Biotechnology, Dallas, TX, USA) as a negative control. The cell lysates were then added to theantibody-bound Protein G Dynabeads and rotated at 4 ◦ C for 2 h. The beads were washed three times
with the lysis buffer. To elute the bound proteins, 60 µL of 2 × SDS-PAGE sample buffer was added,
and the beads were boiled for 5 min.
3. Results
3.1. RIG-I and LGP2 Repress shRNA-Induced RNAi in Human Cells
LGP2, one of the RLRs, represses RNA silencing in human cells by preventing the function of
TRBP by direct interaction [34]. To investigate the contribution of other RLRs, RIG-I and MDA5, for the
regulation of RNA silencing, we performed shRNA-induced RNAi activity assay in human HeLa
cells knocked-down each gene by each of specific siRNAs (siAGO2, siRIG-I, siMDA5, and siLGP2)(Figure 1A). AGO2 is an essential protein in the RNAi pathway and used as a positive control [23,31,32].
The siRNA against GFP was used as a control siRNA (siCont.). The mRNA level of AGO2 decreased
to 40–50% with siAGO2 transfection, and those of all the other genes decreased to 20–40% (Figure S1),and Western blot showed that the protein levels were substantially repressed with or without IFN
treatment (Figure 1B). IFN treatment increased the protein levels of RLRs (RIG-I, MDA5, and LGP2),
but not RNAi factor (AGO2) and control (tubulin) (Figure 1B), as reported previously [34]. We thenperformed a luciferase reporter assay to measure RNAi activity. RNAi activity was measured with aluciferase reporter assay, in which a plasmid expressing shRNA against the firefly luciferase gene wasGenes 2018, 9, 511

5 of 13transfected into the cells with the pGL3-control plasmid expressing the firefly luciferase gene, and thepRL-SV40 plasmid expressing the Renilla luciferase gene as an internal control for normalization.
RNAi activity was shown as the relative luciferase activity (firefly luciferase activity/Renilla luciferase
activity) compared to the control cells transfected with a plasmid expressing shRNA against GFP(shNeg., gray bars in Figure 1C). First, IFN treatment repressed RNAi activities (compare black barof IFN+ with that of IFN− in Figure 1C), as reported previously [34,42]. Knockdown of AGO2
repressed RNAi activity regardless IFN treatment when compared to control cells (compare blackand red bar in each panel of Figure 1C), indicating that AGO2 positively regulates RNAi irrespectiveof IFN treatment (Figure 1C). Contrary to the knockdown of AGO2, that of RIG-I, LGP2, or RLRs
(simultaneous knockdown of RIG-I, MDA5, and LGP2) in IFN-treated HeLa cells increased RNAi
activity. However, the knockdown of MDA5 did not increase RNAi activity. No significant effect wasobserved in the cells knocked down RIG-I, MDA5, LGP2, or RLRs in the absence of IFN treatment(Figure 1C). Thus, upregulation of RIG-I expression by IFN treatment also repress RNAi activity,in addition to LGP2 (Figure 1C).

Figure 1. Retinoic acid-inducible gene I (RIG-I) and laboratory of genetics and physiology 2(LGP2) repress short hairpin RNA (shRNA)-induced RNA interference (RNAi) in human HeLa cells.
(A) Experimental scheme of shRNA-induced RNAi activity assay. Plasmids encoding shRNA against
GFP (shNeg.) or firefly luciferase (shLuc) was transfected with plasmids encoding firefly luciferase andRenilla luciferase into the cells knocked down each gene by small interfering RNA (siRNA) against
either of Argonaute 2 (AGO2), RIG-I, melanoma-differentiation-associated gene 5 (MDA5), LGP2,
or RIG-I like receptors (RLRs) (mixture of siRNAs against RIG-I, MDA5, LGP2). (B) Western blotof AGO2, RIG-I, MDA5, LGP2, and control tubulin in interferon (IFN) non-treated or treated HeLa
cells following transfection of specific siRNA against AGO2, RIG-I, MDA5, LGP2, or RLRs. siRNAagainst GFP was used as control (siCont.). (C) Luciferase reporter assay in wild-type HeLa cells with orwithout IFN. Relative luciferase (luc) activities compared with shNeg. transfection are shown. siRNAagainst each gene was transfected one day ahead of transfection of shLuc to abolish RNA-induced
silencing complex (RISC) saturation. Representative data from two biological repeats, with mean andstandard deviation of technical triplicates. P-values were determined by the Student’s t-test (* p < 0.05).

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6 of 133.2. The Repression of shRNA-Induced RNAi by RIG-I Is TRBP-Dependent
We recently reported that LGP2 represses RNA silencing activity by interacting with the
RNA silencing enhancer, TRBP, which recruits pre-miRNA into Dicer [34]. Our previous
immunoprecipitation assay revealed that only LGP2, but not RIG-I or MDA5, interacts with
TRBP [34]. However, shRNA-induced RNAi activity assay (Figure 1C) showed that RIG-I also represses
shRNA-induced RNAi.
RIG-I and LGP2 have been shown to bind short virus dsRNAs, while MDA5 does not [8].
Thus, an increase in the amount of RIG-I may sequestrate cellular shRNA and disturb RNAi. An EMSA
was performed to examine the interaction of recombinant RLR proteins with 50 -radiolabeled siRNA.
Both RIG-I and LGP2 bound the siRNA, while MDA5 did not (Figure 2A). Thus, one possible reasonof the repression of RNAi by RIG-I is thought to be a result of siRNA sequestration by these proteins.
To examine whether RNA sequestration is major effect of the repression of RNAi by RIG-I,
shRNA-induced RNAi activity assay was performed by the same procedure with Figure 1 in RIG-I−/− ,LGP2−/− , and TRBP−/− cells [34] (Figure S2) established by the clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) genome engineering method [43]. In RIG-I−/−cells, knockdown of LGP2 enhanced RNAi activity significantly, suggesting that RIG-I is not necessary
for inhibition of RNAi by LGP2, probably because LGP2 directly interact with TRBP (Figure 2B).
However, RIG-I knockdown also enhanced RNAi activity in LGP2−/− cells at marginal but significant
level (Figure 2C). TRBP interacts with LGP2 but not RIG-I or MDA5 [34,35], suggesting that RNA
sequestration by RIG-I is a possible mechanism by which RIG-I inhibits RNAi. Since TRBP is an
RNA silencing enhaner, TRBP−/− cells are assumed to exhibit weaker RNAi activity than wild-type
cells, but able to knock down target gene by each siRNA (Figure S3). The knockdown of AGO2
in TRBP−/− cells decreased RNAi activity compared with that of control cells, but knockdown of
RIG-I or LGP2 showed no or little effect (Figure 2D). Our previous report revealed that TRBP canbind a limited fraction of miRNAs, whose stem region has tight base-pairing except for the centerregion, and TRBP can bind to the shRNA used in this experiment [34]. No significant effects wereobserved by knockdown of RIG-I or LGP2 in TRBP−/− cells, probably because RIG-I and LGP2 alsobind to same siRNA (Figure 2A). Thus, the RNA sequestration by RIG-I or LGP2 may not affect RNAi
activity in TRBP-KO cells, because RIG-I, LGP2, and TRBP bind to the same shRNA used in this study.
These results suggest that RNA sequestration by RIG-I and LGP2 may exhibit marginal effects on RNAi
activity in wild type cells, although LGP2–TRBP interaction might be the major inhibitory mechanismof RNAi.
3.3. RIG-I Interacts with TRBP Indirectly through LGP2
Our results indicate that RIG-I is also able to inhibit shRNA-induced RNAi (Figures 1C and 2D)
without interacting with TRBP directly [34], although RNA sequestration by RIG-I may exhibit themarginal effect on the RNAi activity. However, since LGP2 is able to bind TRBP, it was considered that
RIG-I regulates RNA silencing activity by indirect interaction with TRBP through LGP2. Therefore,
we investigated whether RIG-I interacts with LGP2 or not in human embryonic kidney 293T cells(HEK293T). FLAG-TRBP, FLAG-RIG-I or FLAG-MDA5 was transfected into the cells with LGP2-Myc
and immunoprecipitation was carried out. The results revealed that both RIG-I and MDA5 are able tointeract with LGP2, as well as TRBP (Figure 3A).
Next, to examine the possible indirect interaction of RIG-I with TRBP, we investigated whether
TRBP, LGP2, and RIG-I can form triplet complex by immunoprecipitation assay using the expression
plasmid of each gene in HEK293T cells (Figure 3B). At first, expression plasmids of TRBP-Myc
and LGP2-FLAG were co-transfected with the increased concentrations of FLAG-RIG-I plasmid
into the cells. Immunoprecipitation of TRBP protein by anti-Myc antibody revealed that TRBP
certainly interacted with LGP2, but also interacted with RIG-I weakly at the increased concentration of
FLAG-RIG-I, which can interact with LGP2 but not with TRBP (Figure 3B). Furthermore, FLAG-RIG-I
and LGP2-Myc plasmids were co-transfected with the increased concentrations of TRBP-Myc plasmid,

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7 of 13and immunoprecipitation of RIG-I protein by anti-FLAG antibody showed that RIG-I interacted with
LGP2, and with TRBP weakly at the increased concentration of TRBP-Myc, which can interact withLGP2 but not with RIG-I (Figure 3C). These results suggest that TRBP, LGP2, and RIG-I form protein
complex via LGP2, when their expression levels are sufficiently increased.

Figure 2. The repression of shRNA-induced RNAi by RIG-I is TAR-RNA binding protein(TRBP)-dependent. (A) electrophoresis mobility shift assay (EMSA) of recombinant RLR proteinsand siRNA against firefly luciferase (siLuc-36). Each of the purified recombinant RLR proteins and
32 P-labeled siLuc-36 were incubated and electrophoresed on a 9% polyacrylamide gel. (B) Luciferase
reporter assay in RIG-I−/− HeLa cells with IFN. (C) Luciferase reporter assay in LGP2−/− HeLa cellswith IFN. (D) Luciferase reporter assay in TRBP−/− HeLa cells with IFN. Representative data from twobiological repeats, with mean and standard deviation of technical triplicates. P-values were determinedby the Student’s t-test (* p < 0.05).

Furthermore, to examine the interaction of TRBP and LGP2 complex with MDA5, the expression
plasmids of FLAG-MDA5 and TRBP-Myc were co-transfected with the increased concentrations of
LGP2-Myc plasmid into HEK293T cells. Immunoprecipitation of MDA5 by anti-FLAG antibody
showed that MDA5 certainly interacted with LGP2, but not with TRBP at the any concentration of
LGP2-Myc (Figure 3D). The expression plasmids of TRBP-Myc and LGP2-FLAG were co-transfectedGenes 2018, 9, 511

8 of 13with the increased concentrations of FLAG-MDA5 plasmid into the cells, and immunoprecipitation of
TRBP by anti-Myc antibody showed that TRBP interacted with LGP2, but not with MDA5 at the any
concentration of FLAG-MDA5 (Figure 3E). These results suggest that two independent complexes areformed: one consists of MDA5 and LGP2, and the other consists of TRBP and LGP2. Our results ofimmunoprecipitation in HEK293T cells using overexpressed proteins proposed the possibility that
RIG-I may repress shRNA-induced RNAi by inhibiting TRBP function through LGP2, but MDA5
does not.

Figure 3. Cont.

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Figure 3. RIG-I interacts with TRBP indirectly through LGP2. (A) Immunoprecipitation using
HEK293T cells expressing FLAG-TRBP, FLAG-RIG-I, FLAG-MDA5, or FLAG-EGFP with LGP2-Myc
protein. TRBP, RIG-I, MDA5, and enhanced green fluorescent protein (EGFP) proteins were
immunoprecipitated with anti-FLAG antibody, respectively. LGP2 was detected with anti-Myc antibody.
TRBP and EGFP were used as positive and negative controls, respectively. (B) Immunoprecipitation
using cells expressing TRBP-Myc with LGP2-FLAG, and different concentrations of FLAG-RIG-I.
Anti-Myc antibody was used for immunoprecipitation of TRBP, and LGP2 and RIG-I were detectedwith anti-FLAG antibody. The signal intensities of LGP2 of immunoprecipitate and input,
and immunoprecipitated RIG-I were measured using ImageQuant TL (GE Healthcare). The relative
ratio of LGP2 signal intensity (immunoprecipitate/input) compared to the control sample without
FLAG-RIG-I transfection is shown in pink, and the relative ratio of RIG-I signal intensity compared tothe cells transfected with FLAG-RIG-I (6 µg) is shown in blue. (C) Immunoprecipitation using cells
expressing FLAG-RIG-I with LGP2-Myc, and different concentrations of TRBP-Myc. The relative
ratio of LGP2 signal intensity is shown in pink. The relative ratio of TRBP signal intensity isshown in green. (D) Immunoprecipitation using cells expressing FLAG-MDA5 with TRBP-Myc,
and different concentrations of LGP2-Myc. (E) Immunoprecipitation using cells expressing TRBP-Myc
with LGP2-FLAG, and different concentrations of FLAG-MDA5. (B–E) Total amount of empty plasmid
and TRBP or each RLR construct was unified at 6 µg in each sample. The presumed results of eachprotein interaction were shown below each panel.

4. Discussion
We recently reported that one of the RLRs, LGP2, interacts with the RNA silencing enhancer, TRBP,
to repress its function [34]. TRBP functions to recruit TRBP-bound pre-miRNA into Dicer, resulting
enhancement of the miRNA maturation [24,25], and TRBP has greatly higher dsRNA binding affinity
compared with Dicer [44], indicating the importance of pre-miRNA recruitment by TRBP in vivo.
LGP2 interacts with dsRNA-binding sites of TRBP competitively with dsRNA [34]. This competitive
binding inhibits the recruitment of pre-miRNA, the miRNA maturation, and subsequent RNA silencing
activity of TRBP-bound miRNAs [34]. In this report, we examined the contribution of other RLRs,
RIG-I and MDA5, for the regulation of RNA silencing. We showed that IFN treatment attenuated RNAi
activity in human cells, and knockdown of RIG-I but not MDA5 enhanced RNAi activity (Figure 1C).
IFN treatment increased RLR protein expression (Figure 1B), and upregulated RIG-I repressed RNAi
activity by potentiating the effect of LGP2. This may indicate that RIG-I functions to stabilize theinteraction between LGP2 and TRBP, although it is unclear why RIG-I depletion enhances RNAi morethan LGP2 depletion. Furthermore, we examined the possibility of RNA sequestration by RIG-I for
the repression of RNAi. EMSA showed that recombinant RIG-I and LGP2 proteins bound to siRNA,
respectively (Figure 2A), as well as TRBP [36]. The shRNA-induced RNAi activity assay in LGP2−/−
cells showed that RIG-I knockdown enhanced RNAi activity significantly at marginal level (Figure 2C),
suggesting that RNA sequestration by RIG-I is a possible and not negligible mechanism by which RIG-I

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inhibits RNAi. In TRBP−/− cells, RIG-I did not repress shRNA-induced RNAi (Figure 2D). TRBP canbind a limited fraction of miRNAs, and TRBP can bind to the shRNA used in this experiment [34].
Since RIG-I and LGP2 also bind to same siRNA (Figure 2A), no significant effects by knockdown of
RIG-I or LGP2 in TRBP−/− cells might be observed. Thus, RNA sequestration by RIG-I and LGP2 may
exhibit modest effects on RNAi activity.
Immunoprecipitation assays in HEK293T cells using expression plasmids revealed that RIG-I
interacts with the RNA silencing enhancer, TRBP, indirectly through LGP2. In our previous report,
we performed immunoprecipitation assay in HeLa cells to detect endogenous RLR proteins interactingwith TRBP, and identified that LGP2 interacts with TRBP directly [34]. In this report, we transfected
the expression plasmids encoding RIG-I, MDA5, and LGP2 into HEK293T cells to increase their protein
levels sufficiently to detect the indirect interaction with TRBP clearly. Figure 3A shows that both
RIG-I and MDA5 can interact with LGP2, independently. Furthermore, LGP2 interacted with both
TRBP and RIG-I at once, but did not interact with TRBP and MDA5 simultaneously (Figure 3B–E).
Such distinguishable interactions of RIG-I and MDA5 with TRBP-LGP2 may explain the mechanismthat knockdown of RIG-I, but not MDA5, increased shRNA-induced RNAi activity (Figure 1C).
During viral infection, RIG-I and MDA5 recognize different types of RNA viruses to produce antiviralcytokines. This may indicate that a specific type of virus can repress RNA silencing activity. However,
it is unclear how the inhibitory effect on RNAi is related to the anti-viral response. Further studies areneeded to examine whether RIG-I represses endogenous miRNA-mediated RNA silencing such as
LGP2 and identify their regulated genes.
In summary, one of viral sensor proteins, RIG-I, repressed shRNA-induced RNAi and theinteraction with TRBP-LGP2 may have major effect on the repression of RNAi activity. Our finding
suggests that RIG-I may function as an RNAi modulator in collaboration with LGP2 and partiallythrough RNA sequestration. These results consolidated our previous report showing the crosstalk
between RNA silencing and RLR signaling in mammalian cells.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/9/10/511/s1,
Figure S1: Relative mRNA level of AGO2, RIG-I, MDA5, or LGP2 in IFN non-treated or treated cells, Figure S2:Generation of RIG-I knockout HeLa cells (RIG-I− /− ), Figure S3: Confirmation of knockdown at protein level bytransfection of siAGO2, siRIG-I, siMDA5, or siLGP2 in TRBP− /− cells, Table S1: siRNA sequences used for geneknockdown and EMSA, Table S2: PCR primers used in this study for plasmid construction, Table S3: PCR primers
used in this study for qRT-PCR.
Author Contributions: T.T. and K.U.-T. designed the study. T.T., Y.N., K.O., M.Y., and K.U.-T. discussed themethods, results, and the experimental plans. T.T., Y.N., and K.O. performed the experiments. The manuscript
was drafted by T.T. and K.U.-T. T.T., M.Y., and K.U.-T. reviewed the manuscript. All authors read and approved
the final manuscript.
Funding: This work was supported by the grants from the Ministry of Education, Culture, Sports, Science andTechnology of Japan (21310123, 21115004, 15H04319, 16H14640, 221S0002, and 16H06279 to K.U.-T.; and 15K19124
and 18K15178 to T.T.], the grants provided from the Ichiro Kanehara Foundation, the Inamori Foundation, theUehara Memorial Foundation, and Japan Science and Technology Agency to T.T., Suzuken Memorial Foundation
and Japan Health & Research Institute to K.U.-T., and Joint Usage/Research Program of Medical Mycology
Research Center, Chiba University (14-14, 15-16, 16-1, 17-15, 18-11) to T.T., K.O., M.Y. and K.U.-T.
Acknowledgments: We thank T. Fujita for kindly providing RIG-I and MDA5 expression plasmids.
Conflicts of Interest: The authors declare no conflict of interest.

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