Article

The RNA Binding Protein Mex-3B Is Required for IL33 Induction in the Development of Allergic Airway
Inflammation
Graphical Abstract

Authors
Yusuke Yamazumi, Oh Sasaki,
Mitsuru Imamura, ..., Makoto Dohi,
Shigeo Koyasu, Tetsu Akiyama

Correspondence
akiyama@iam.u-tokyo.ac.jp

In Brief
Yamazumi et al. show that Mex-3Bmediated post-transcriptional
upregulation of IL-33 is required for thedevelopment of allergic airway
inflammation. Inhalation of an antisense
oligonucleotide targeting Mex-3B
suppresses allergic airway inflammation,suggesting that Mex-3B could be a
promising molecular target for thetreatment of allergic asthma.

Highlightsdd

Mex-3B induces IL-33 in the development of allergic airway
inflammation
Mex3b / mice develop less airway inflammation due toreduced induction of IL-33

d

Mex-3B upregulates IL-33 expression by inhibiting miR487b-3p-mediated repressiondInhalation of an antisense oligo targeting Mex-3B suppresses
airway inflammation

Yamazumi et al., 2016, Cell Reports 16, 2456–2471
August 30, 2016 ª 2016 The Author(s).
http://dx.doi.org/10.1016/j.celrep.2016.07.062

Cell Reports

ArticleThe RNA Binding Protein Mex-3B Is Required
for IL-33 Induction in the Developmentof Allergic Airway Inflammation
Yusuke Yamazumi,1 Oh Sasaki,2 Mitsuru Imamura,2 Takeaki Oda,1 Yoko Ohno,1 Yumi Shiozaki-Sato,1 Shigenori Nagai,3
Saki Suyama,1 Yuki Kamoshida,1 Kosuke Funato,1 Teruhito Yasui,4 Hitoshi Kikutani,4 Kazuhiko Yamamoto,2Makoto Dohi,2,5 Shigeo Koyasu,6 and Tetsu Akiyama1,7,*
1Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-0032, Japan
2Department of Allergy and Rheumatology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655,
Japan
3Department of Molecular Immunology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
4Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita,
Osaka 565-0871, Japan
5Institute of Respiratory Immunology, Shibuya Clinic for Respiratory Diseases and Allergology, Tokyo 150-0002, Japan
6Laboratory for Immune Cell Systems, RIKEN Research Center for Integrative Medical Sciences (IMS), 1-7-22 Suehiro-cho, Tsurumi-ku,
Yokohama 230-0045, Japan
7Lead Contact*Correspondence: akiyama@iam.u-tokyo.ac.jp
http://dx.doi.org/10.1016/j.celrep.2016.07.062

SUMMARY

Allergic airway inflammation is one of the primaryfeatures of allergic asthma. Interleukin-33 (IL-33)
is recognized as a key pro-inflammatory cytokine
that mediates allergic airway inflammation, and its
expression is elevated in this condition, but little is
known about the regulatory mechanisms underlying
IL-33 induction. Here, we show that the RNA binding
protein Mex-3B plays a critical role in the induction of
IL-33 in the development of allergic airway inflammation. We generated Mex3b/ mice and found thatthey develop significantly less airway inflammation
than wild-type mice due to reduced induction of IL33. Furthermore, we show that Mex-3B directly upregulates IL-33 expression by inhibiting miR-487b-3pmediated repression of IL-33. Moreover, we showthat inhalation of an antisense oligonucleotide targeting Mex-3B suppresses allergic airway inflammation. Our data identify a signaling pathway that
post-transcriptionally regulates IL-33 expression
and suggest that Mex-3B could be a promising molecular target for the treatment of allergic asthma.
INTRODUCTION
Allergic asthma is a chronic inflammatory disease of the lungs
that affects nearly 300 million people worldwide. Allergic airway
inflammation is one of the primary features of allergic asthma
and is generally considered to be mediated by a T helper type2 (Th2) response (Kim et al., 2010). Leucocytes have long

been focused as the key players in the development of allergic
airway inflammation. However, there is increasing evidence thatepithelial cells act as active immune regulators that produce
inflammatory factors, such as TSLP and interleukin-33 (IL-33)(Liew et al., 2010; Ziegler and Artis, 2010). IL-33 is a memberof the IL-1 family, which plays important roles in type 2 innate
immunity and human asthma (Cayrol and Girard, 2014). It
has been shown that mice deficient for IL-33 are resistantto allergen-induced airway inflammation (Oboki et al., 2010).
Furthermore, IL-33 has been identified as a susceptibility genefor human asthma (Gudbjartsson et al., 2009; Moffatt et al.,2010). In addition, its expression in epithelial cells is higher
in patients with allergic asthma than in healthy individuals(Préfontaine et al., 2010), and upregulation of IL-33 expression
is observed in an experimental asthma model (Louten et al.,2011). Although several studies have shown that IL-33 expression is induced upon pro-inflammatory cytokine stimulation inseveral cell types (Masamune et al., 2010; Sponheim et al.,2010), the importance of IL-33 induction in the developmentof allergic airway inflammation, and the mechanisms underlying
this induction are not well understood.
MicroRNAs (miRNAs) are small non-coding RNAs that canregulate gene expression and have been shown to play a rolein various pathologies, including inflammation and cancer.
miRNAs interact with Argonaute (Ago) and other proteins toform RNA-induced silencing complexes (RISCs), and guidethese RISCs to 30 UTRs of target RNAs via sequence-specificbinding (Bartel, 2009; Fabian et al., 2010). This results in thedestabilization of the target RNA and inhibition of translation.
RNA binding proteins can also interact with 30 UTRs and regulate
gene expression (Filipowicz et al., 2008; Kedde and Agami,2008). Recently, several groups have obtained evidence thatmiRNAs and RNA binding proteins can co-operate at common

2456 Cell Reports 16, 2456–2471, August 30, 2016 ª 2016 The Author(s).
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

target 30 UTRs under certain conditions (Bhattacharyya et al.,2006; Ray et al., 2009).
Mex-3B, a member of the Mex-3 family, contains two K homology (KH)-type RNA-recognition domains and a RING finger
domain (Buchet-Poyau et al., 2007). It has been shown that thesefamily members associate with specific mRNAs and regulate
expression of their proteins (Cano et al., 2012; Donnini et al.,
2004; Pereira et al., 2013). Mex-3D (also known as TINO or
RKHD1) has been shown to destabilize BCL2 mRNA by binding
to an AU-rich element in its 30 UTR (Donnini et al., 2004). Mex-3C
regulates HLA-A2 levels through its mRNA 30 UTR (Cano et al.,2012). Mex-3A has been shown to repress CDX2 expression
post-transcriptionally through binding to the CDX2 30 UTR (Pereira et al., 2013). Mex3b-deficient mice exhibit defects in Sertoli
cells and have low fertility (Le Borgne et al., 2014). However, thephysiological role of post-transcriptional regulation of target
genes by Mex-3B has not been elucidated.
In this study, we generated Mex3b/ mice and used them inan experimental model of allergic airway inflammation. We showthat Mex3b/ mice develop significantly less airway inflammation, due to reduced induction of IL-33. We also demonstratethat Mex-3B binds to the IL-33 30 UTR and upregulates IL-33
expression by inhibiting miR-487b-3p-mediated repression of
IL-33. Furthermore, we show that inhalation of a locked nucleic
acid (LNA)-modified antisense oligonucleotide targeting Mex3B (LNA-anti-Mex-3B) suppresses the development of allergic
airway inflammation.

Mice Deficient in Mex3b Have Normal Phenotype
To gain insight into Mex-3B function in vivo, we generated
Mex3b/ mice through homologous recombination in mouseembryonic stem cells (Figures S1A–S1E). Homozygous Mex3b
mutant animals were born in a Mendelian ratio (25% of progeny), showing no evidence of embryonic lethality. Mex3b/
newborns were indistinguishable from wild-type littermates
except that they were smaller and had reduced body weight(Figure S1F). Consistent with a previous report (Le Borgne
et al., 2014), Mex3b/ mice were fertile, but fertility is low.
Macroscopic and histological examination did not reveal any
morphological abnormalities in the major organs of young
adult mice. In addition, Mex3b/ mice showed no detectable
abnormalities in the development of B cells in the spleen or
T cells in the thymus (Figure S1G).

cytic infiltration and mucus secretion (Figure 1B) and decreased
numbers of bronchoalveolar lavage (BAL) eosinophils (Figure 1C)
in response to OVA challenge. The Th2-type cytokines, IL-4,
IL-5, and IL-13, were less efficiently induced in the lungs of
Mex3b/ mice (Figure 1D). By contrast, the Th1-type cytokine
interferon-g (IFN)-g was induced normally. We also examined
the number of Th2 cells in bronchial lymph nodes by measuring
intracellular cytokine levels of lung bronchial lymph node cells
stimulated ex vivo with leukocyte activation cocktail. We
observed decreased percentages of IL-4-positive CD4+ cells inprimary cultured bronchial lymph node cells from Mex3b/
mice (Figure S2A). These results suggest that Mex-3B is required
for the development of allergic airway inflammation.
Th2 cells are known to play critical roles in the pathogenesis ofallergic airway inflammation by producing a variety of cytokines
such as IL-4, IL-5, IL-9, and IL-13 (Herrick and Bottomly, 2003).
In addition to Th2 cells, it has recently been shown that Th1, regulatory T (Treg), and Th17 cells play important roles in allergic
airway inflammation (Lloyd and Hessel, 2010). To understandhow Mex-3B regulates the induction of allergic airway inflammation, we first examined T cell differentiation in Mex3b/ mice.
Effector and regulatory T cells were generated from wild-type
and Mex3b/ CD4+CD62L+ naive T cells by standard methods.
We found that the percentage of each T cell subset generated
was very similar regardless of their Mex3b status (Figure S2B).
Furthermore, we stimulated CD4+ T cells with anti-CD3/CD28
antibody in vitro and examined the expression of Mex-3B andthe other family members. IFN-g was used as a marker ofT cell activation. We found that stimulation with anti-CD3/CD28
antibody resulted in reduced expression of only Mex-3B (Figure S2C). We next examined antigen-induced immune responses in the spleens. When splenocytes from OVA-sensitizedmice were cultured with OVA, similar levels of IL-5 were secreted
into the culture media by wild-type and Mex3b/ splenocytes(Figure S2D). These results suggest that immune cell functionin the spleen is comparable between wild-type and Mex3b/
mice.
To further clarify the role of Mex-3B in hematopoietic cells,
we performed bone marrow transplantation experiments. BAL
fluid analysis revealed that Mex3b/ mice transplanted withbone marrow cells from wild-type mice had less inflammation
than wild-type mice transplanted with bone marrow cells from
Mex3b/ mice (Figures S2E and S2F). These results exclude
the possibility that Mex-3B in hematopoietic cells plays a criticalrole in the development of allergic airway inflammation.

Mex-3B Is Required for the Ovalbumin-Induced Allergic
Airway Inflammation
It has previously been reported that Mex-3B is relatively highly
expressed in human lung and thymus (Buchet-Poyau et al.,2007). Consistent with these results, we found that Mex-3B isalso highly expressed in mouse lung and thymus (Y.Y., unpublished data). We therefore speculated that Mex-3B could playa role in the immune system in the lung. We examined this possibility using an experimental model of allergic airway inflammation (Figure 1A). Mice were sensitized with ovalbumin (OVA)/alum
and then challenged with nebulized OVA or PBS. Compared towild-type mice, Mex3b/ mice exhibited decreased lung leuko-

IL-33 Induction Is Suppressed in Mex3b-Deficient Mice
Recent studies have shown that airway epithelial cells play critical roles in the regulation of airway inflammation: airway epithelial cells influence the function of immune-competent cells, suchas dendritic cells (Hammad and Lambrecht, 2008), mast cells(Allakhverdi et al., 2007), and Th2 cells (Wang et al., 2007) by producing many inflammatory factors in response to allergen exposure. We therefore examined the expression of Mex-3B in lungtissues from control and OVA-challenged mice. We separated
airway epithelial and non-epithelial cells using anti-CD45 antibody and performed qRT-PCR analysis. IL-13 and SP-C wereused as markers of inflammation and airway epithelial cells,

RESULTS

Cell Reports 16, 2456–2471, August 30, 2016 2457

Figure 1. Mex-3B Is Critical for the Development of Airway Inflammation(A) Scheme of the experimental asthma model. Mex3b+/+ and Mex3b/ mice were sensitized with OVA/alum on days 0 and 11. The mice were nebulized with
OVA or PBS from day 18 to 20 and analyzed on day 21.
(B) OVA-induced infiltration of eosinophils into the peribronchial area and mucus hypersecretion by bronchial epithelial cells. (left) H&E staining. Scale bar,
100 mm. (right) Periodic acid-Schiff staining. Scale bar, 15 mm.
(C) Bronchoalveolar lavage (BAL) fluid analyses were performed. Leukocytes were identified by morphological criteria.
(D) Cytokine concentrations in the lungs of OVA-challenged or control mice. Lung homogenates were assayed for cytokine concentrations by ELISA. Data arerepresented as mean ± SEM (n = 3–7). *p < 0.05, **p < 0.01.
See also Figures S1–S3.

2458 Cell Reports 16, 2456–2471, August 30, 2016

Figure 2. Mex-3B Is Required for the Induction of IL-33 Expression in the Developmentof Allergic Airway Inflammation(A) The levels of inflammatory factor mRNAs in
OVA-inflamed lungs of Mex3b+/+ and Mex3b/
mice.
(B) Immunoblot analysis of IL-33 protein in control
or OVA-inflamed lungs.
(C) Lung sections were stained with anti-IL-33
antibody and counterstained with hematoxylin.
Scale bar, 20 mm. Data are represented as mean ±SEM (n = 3–7). *p < 0.05, **p < 0.01.
See also Figure S4.

ure 2C). Following OVA challenge, we
detected strong expression of IL-33 in
the nuclei of alveolar epithelial cells fromwild-type, but not Mex3b/ mice (Figure 2C). By contrast, IL-33 was induced
to similar levels in the cytoplasm of alveolar macrophages from either wild-type
or Mex3b/ mice (Figure S4A). Furthermore, the levels of IL-33 mRNA induced
by LPS in OVA-sensitized or unsensitized alveolar macrophages were similar
in wild-type and Mex3b/ mice (Figure S4B). These results suggest thatMex-3B is required for the inflammationinduced upregulation of IL-33 in alveolar
epithelial cells.

respectively. We found that Mex-3B was expressed in bothairway epithelial and non-epithelial cells at similar levels andwas upregulated by OVA challenge (Figure S3). We also foundthat the other Mex family genes, Mex-3A, -3C, and -3D, werehighly expressed in airway epithelial cells compared with nonepithelial cells, and that only Mex-3A was upregulated by OVA
challenge in airway epithelial cells. These results suggest thatMex-3B plays a role in airway epithelial cells in the developmentof allergic airway inflammation.
To investigate the role of Mex-3B in inflammatory factor production from airway epithelial cells, we examined the mRNAlevels of epithelial cell-derived inflammatory factors in the lungtissues of OVA-challenged wild-type and Mex3b/ mice. Amongthe inflammatory factors examined, IL-33 and GM-CSF mRNAs
were found to be less efficiently induced in Mex3b/ comparedto wild-type lung (Figure 2A). Similarly, IL-33 protein was not
significantly induced in the inflamed lungs of Mex3b/ micecompared with wild-type mice (Figure 2B). In agreement with aprevious report (Louten et al., 2011), immunostaining with antiIL-33 antibody revealed that IL-33 was localized to the nuclei of
alveolar epithelial cells of both wild-type and Mex3b/ mice (Fig-Mex-3B Deficiency Does Not
Significantly Affect IL-33-Induced
Airway Inflammation
IL-33 is a central player in the development of allergic inflammation. IL-33 stimulates Th2 cells (Schmitz et al., 2005), mast cells (Iikura et al.,2007), and natural helper cells (Moro et al., 2010). To clarify
whether decreased IL-33 production in Mex3b/ mice is
responsible for the impaired development of airway inflammation, we examined the effects of intranasal administration of IL33 on lung inflammation in wild-type and Mex3b/ mice (Figure S4C). Administration of IL-33 for 4 consecutive days resulted
in airway inflammation in both wild-type and Mex3b/ mice,similar to that reported by Kondo et al. (2008), and was manifested by an increase in the numbers of eosinophils (Figure S4D),
induction of Th2 cytokines (Figure S4E), and epithelial thickening
and leukocytic infiltration (Figure S4F). Thus, in contrast to OVAinduced airway inflammation, IL-33-induced inflammation is notaffected by Mex3b status. These results suggest that Mex3b/
mice develop less airway inflammation than wild-type littermates
due to reduced induction of IL-33.
Mex-3B Promotes IL-33 Expression Posttranscriptionally
We next investigated the mechanisms by which Mex-3B induces
IL-33 expression using the transformed murine respiratory

Cell Reports 16, 2456–2471, August 30, 2016 2459

(legend on next page)2460 Cell Reports 16, 2456–2471, August 30, 2016

epithelial cell-derived cell line MLE-15 (Wikenheiser et al., 1993).
We first examined whether IL-33 expression in MLE-15 cells is
also regulated by Mex-3B. We found that knockdown of Mex3B by small interfering RNA (siRNA) caused a decrease in both
IL-33 mRNA and protein expression (Figures 3A and 3B). We
therefore set out to examine whether Mex-3B directly regulates
IL-33 expression using MLE-15 cells.
Since Mex-3B is a KH-type RNA binding protein, we investigated whether Mex-3B is associated with the IL-33 mRNA. We
performed RNA immunoprecipitation assays: lysates preparedfrom MLE-15 cells were subjected to immunoprecipitation with
anti-Mex-3B antibody and the IL-33 mRNA associated with the
immunoprecipitates was detected by qRT-PCR analysis. We
found that Mex-3B is associated with IL-33 mRNA, but not significantly with HPRT1 mRNA (Figure 3C). To examine the role of theKH domain in Mex-3B binding to IL-33 mRNA, we transfected
FLAG-tagged wild-type or KH domain mutant Mex-3B into thealveolar epithelial cell-derived adenocarcinoma cell line A549
(Lieber et al., 1976) and performed RNA immunoprecipitation assays. We found that Mex-3B is associated with IL-33 mRNA, but
not with GAPDH mRNA (Figure 3D). By contrast, we found thatMex-3B-mutKH did not co-precipitate with the IL-33 mRNA.
Mex-3B-mutKH is mutated in key residues involved in RNA binding, i.e., Gly-83 and Gly-177 in the KH domains, which are replaced with Asp. Thus, Mex-3B may associate with the IL-33
mRNA through its KH domains.
An array of RNA binding proteins recognizes the 30 UTR oftarget genes and exerts post-transcriptional control (Khabar,
2010). To examine whether Mex-3B binds to the IL-33 30
UTR, we performed RNA binding protein pull-down assays.
Lysates prepared from A549 cells transfected with FLAGtagged Mex-3B were incubated with biotin-labeled IL-33 30
UTR and subjected to precipitation with streptavidin-Dynabeads. Immunoblotting with anti-FLAG antibody revealed
that Mex-3B co-precipitated with the IL-33 30 UTR, but notwith the GAPDH 30 UTR (Figure 3E). Furthermore, we produced
recombinant Mex-3B fused to glutathione S-transferase (GSTMex-3B) (Figure S5) and examined its ability to interact directlywith the IL-33 30 UTR by RNA binding protein pull-down assays. Immunoblotting of the precipitates with anti-Mex-3B
antibody revealed that GST-Mex-3B directly binds to the IL33 30 UTR (Figure 3F).
To clarify the significance of the association of Mex-3B with
the IL-33 30 UTR, we constructed a reporter vector containing

the IL-33 30 UTR region fused downstream of the luciferase
gene. We found that knockdown of Mex-3B by siRNA caused
a decrease in luciferase activity in MLE-15 cells (Figure 3G).
Furthermore, we found that overexpression of Mex-3B, but notof Mex-3B-mutKH resulted in the enhancement of reporter activity in A549 cells (Figure 3H). These results suggest that Mex-3B
post-transcriptionally activates IL-33 expression through binding to the IL-33 30 UTR.
Identification of a Mex-3B-Responsive Region in the
IL-33 30 UTR
We next investigated which region of the IL-33 30 UTR (region
1–12 in Figures S6A–S6D) is involved in Mex-3B-mediated upregulation of IL-33. We found that luciferase reporters containing
region 9 [30 UTR-9 (nucleotides 1414–1674)] were significantly
upregulated by Mex-3B (Figure S6C). Furthermore, RNA binding
protein pull-down assays revealed that Mex-3B binds 30 UTR-9(Figure S6D). These results suggest that Mex-3B regulates IL33 expression through binding to 30 UTR-9.
Mex-3B Promotes IL-33 Expression by Inhibiting
miR-487b-3p-Mediated Repression
It has been shown that miRNAs suppress the expression oftarget genes by binding to their 30 UTRs (Bartel, 2009). We therefore speculated that the Mex-3B-mediated regulation of IL-33
expression via the 30 UTR might involve an miRNA species.
We therefore searched for potential miRNA binding sites in 30
UTR-9 using the microRNA.org database (John et al., 2004)
and identified seven candidate sites (Figure 4A). Among thesemiRNAs, overexpression of miR-487b-3p most significantly suppressed IL-33 mRNA and protein expression in MLE-15 cells(Figures 4B and 4C). Overexpression of miR-487b-3p also resulted in a decrease in the activity of the luciferase reporters
containing either the entire 30 UTR or 30 UTR-9 (Figure 4D). By
contrast, overexpression of miR-487b-3p did not inhibit the activity of a luciferase reporter containing a mutated miR-487b3p binding site (30 UTR-9-mt487b in Figures 4E and 4F). In addition, this mutant reporter showed enhanced activity in theabsence of Mex-3B. These results suggest that miR-487b-3p
directly regulates IL-33 expression.
We next examined the significance of miR-487b-3p in Mex3B-mediated upregulation of IL-33. We found that knockdownof miR-487b-3p by a locked nucleic-acid-modified antisense
oligonucleotide (LNA-anti-miR487b-3p) (Ørom et al., 2006)

Figure 3. Mex-3B Post-transcriptionally Upregulates IL-33 Expression in the Alveolar Epithelial Cells(A and B) Effects of Mex-3B knockdown on IL-33 mRNA (A) and protein (B) expression in MLE-15 cells.
(C) qRT-PCR analysis of IL-33 mRNA co-immunoprecipitated with rabbit immunoglobulin G (IgG) or anti-Mex-3B antibody in MLE-15 cells. HPRT1 mRNA wasused as a negative control.
(D) qRT-PCR analysis of IL-33 mRNA co-immunoprecipitated with mouse IgG or anti-FLAG-M2 antibody in A549 cells, transfected with control vector, FLAGtagged Mex-3B or FLAG-tagged Mex-3B-mutKH. GAPDH mRNA was used as a negative control.
(E) Immunoblot analysis of exogenously expressed FLAG-tagged Mex-3B protein co-precipitated with the indicated in vitro transcribed biotin-labeled RNAs. (left)
Input. (right) Mex-3B pulled-down with biotin-labeled RNAs. GAPDH 30 UTR was used as a negative control.
(F) Immunoblot analysis of GST-Mex-3B co-precipitated with the indicated in vitro transcribed biotin-labeled RNAs. GAPDH 30 UTR was used as a negative
control.
(G) Luciferase activity of Mex-3B-knockdowned MLE-15 cells transfected with a luciferase reporter fused to the IL-33 30 UTR.
(H) Luciferase activity of A549 cells transfected with a luciferase reporter fused to the IL-33 30 UTR along with control vector, FLAG-tagged Mex-3B or FLAGtagged Mex-3B-mutKH. Data are represented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.
See also Figure S5.

Cell Reports 16, 2456–2471, August 30, 2016 2461

Figure 4. Mex-3B Inhibits the Effect of miR-487b-3p on the IL-33 30 UTR(A) Target sites of conserved miRNAs in IL-33 30 UTR-9 predicted by mirSVR score (http://www.microRNA.org).
(B and C) Effect of miRNA overexpression on IL-33 mRNA (B) and protein (C) expression in MLE-15 cells.
(D) Luciferase activity of MLE-15 cells transfected with each reporter plasmid along with control miRNA or miR-487b-3p.
(E) Schematic representation of wild-type and mutated 30 UTR-9 reporters.
(F) Luciferase activity of MLE-15 cells co-transfected with each reporter plasmid along with control miRNA or miR-487b-3p.
(G) Immunoblot analysis of IL-33 in MLE-15 cells transfected with the indicated anti-miRNAs along with the indicated siRNAs.

(legend continued on next page)2462 Cell Reports 16, 2456–2471, August 30, 2016

resulted in a restoration of IL-33 protein expression in MLE-15
cells in which Mex-3B was knocked down (Figure 4G). In addition, overexpression of Mex-3B restored IL-33 protein expression, as well as the activity of a luciferase reporter containing
30 UTR-9 in cells in which miR-487b-3p was overexpressed(Figures 4H and 4I). We also found that knockdown of Mex3B resulted in a decrease in miR-487b-3p expression (Figure S6E). These results suggest that Mex-3B promotes IL-33
expression by inhibiting miR-487b-3p-mediated suppressionof IL-33.
Mex-3B Inhibits the Interaction of the miR-487b-3pAgo2 Complex with the miR-487b-3p Target Site in the
IL-33 30 UTR
RNA binding proteins such as Dnd1 have been shown to
inhibit miRNA access to their target mRNAs (Kedde et al.,2007). Thus, it is possible that Mex-3B competes with the miR487b-3p-RISC complex for binding to the 30 UTR of IL-33. To
examine this possibility, we performed RNA binding protein
pull-down assays. When miR-487b-3p was transfected into
A549 cells, Ago2 was found to co-precipitate with 30 UTR-9 (Figure 5A). However, co-transfection of Mex-3B with miR-487b-3p
interfered with the co-precipitation of Ago2 and 30 UTR-9 (Figure 5A). These results suggest that Mex-3B inhibits the interaction of the miR-487b-3p-Ago2 complex with the miR-487b-3p
target site in the IL-33 30 UTR.
We therefore examined whether the Mex-3B binding site overlaps with the miR-487b-3p binding site. However, we found thatMex-3B coprecipitated with a mutated 30 UTR, 30 UTR-9-Dseed,in which the miR-487b-3p seed match sequence in 30 UTR-9 hasdeleted (Figures 5B and 5C). Thus, Mex-3B may not directlycompete with the miR-487b-3p-RISC complex for binding to
the 30 UTR of IL-33 but indirectly inhibits their interaction presumably by changing the RNA secondary structure.
As shown in Figure S6C, we found that Mex-3B activated aluciferase reporter containing 30 UTR-9, but not 30 UTR-11 or 30
UTR-12, which are the 50 and 30 halves of 30 UTR-9, respectively.
This may indicate that a region straddling 30 UTR-11 and 30 UTR12 is important for Mex-3B binding. In addition, RNA secondary
structure prediction indicated the presence of a stem-loop structure, within which this region is contained. We therefore constructed a mutated 30 UTR-9, 30 UTR-9-DSL, in which this predicted stem-loop structure (nucleotides 1514–1547) is deleted.
Luciferase assays revealed that Mex-3B did not enhance theactivity of a reporter containing 30 UTR-9-DSL (Figure 5E). Moreover, RNA binding protein pull-down assays revealed that Mex3B coprecipitated only weakly with 30 UTR-9-DSL (Figure 5F).
We also found that Ago2 coprecipitated only weakly with 30
UTR-9-DSL and that miR-487b-3p did not affect this interaction(Figure 5G). Thus, the region containing nucleotides 1514–1547
is required for the interaction of both Mex-3B and the miR487b-3p-Ago2 complex with the IL-33 30 UTR.

miR-487b-3p Is Important for the Development ofAllergic Airway Inflammation
To investigate whether miR-487b-3p plays a role in the negative regulation of allergic airway inflammation, LNA-anti-miR487b-3p was administered into OVA-sensitized wild-type or
Mex3b/ mice according to the protocol shown in Figure 6A.
Cytological analysis of BAL fluid revealed that nebulizationof either LNA-anti-miR-487b-3p or a control LNA antisense
oligonucleotide (LNA-anti-miR-control) had no effect on OVAinduced inflammation in wild-type mice (Figure 6B). By contrast,
nebulization of LNA-anti-miRNA-487b-3p, but not an LNA-antimiR-control, caused an increase in eosinophil infiltration in
Mex3b/ mice challenged with OVA (Figures 6A and 6B). We
also found that nebulization of LNA-anti-miR-487b-3p restored
IL-33 protein levels in Mex3b/ mice (Figure 6C). In addition,
we confirmed that Alexa-488-labeled LNA-anti-miR-487b-3p
administered in a nebulized form accumulated in lung epithelial
cells 24 hr after exposure (Figure S7A). These observations are
consistent with the notion that miR-487b-3p plays an importantrole in the downregulation of IL-33 and thereby suppresses
airway inflammation.
We performed qRT-PCR analysis to measure the approximate copy numbers of miR-487b-3p and IL-33 RNA per cellin the lung tissues of control and OVA-challenged Mex3b+/+and Mex3b/ mice. We used miRNA-505-3p and miRNA875-5p as controls, which are expressed in MLE-15 cells but
do not suppress IL-33 expression. We found that the expression of miR-487b-3p and miR-875-5p was lower than that of
IL-33 mRNA, while the expression of miR-505-3p was similar
to that of IL-33 mRNA (Figure S7B). In addition, we observedthat the expression of miR-487b-3p was similar between control and OVA-challenged mice. These results suggest that the
suppression of IL-33 expression observed in OVA-challenged
mice is not simply the result of altered expression of miR487b-3p.
LNA Antisense Oligonucleotide-Mediated Knockdownof Mex-3B Suppresses Allergic Airway Inflammation
We finally investigated whether administration of an LNA antisense oligonucleotide targeting Mex-3B (LNA-anti-Mex-3B)
could suppress the development of allergic airway inflammation.
We confirmed that overexpression of LNA-anti-Mex-3B resulted
in a decrease in the levels of Mex-3B and IL-33 in MLE-15 cells(Figure 7A). When LNA-anti-Mex-3B was administered into OVAsensitized wild-type mice according to the protocol shown in
Figure 7B, eosinophil infiltration was significantly inhibited(Figure 7C). Immunostaining with anti-IL-33 antibody revealed
that LNA-anti-Mex-3B nebulization caused a decrease in thenumber of cells that were strongly positive for IL-33 (Figure 7D).
These results suggest that suppression of Mex-3B expression
using LNA antisense oligonucleotides suppresses allergic airway
inflammation.

(H) Immunoblot analysis of IL-33 in MLE-15 cells transfected with the indicated miRNAs along with control vector or FLAG-tagged Mex-3B.
(I) Luciferase activity of MLE-15 cells co-transfected with each reporter plasmid and vector or FLAG-tagged Mex-3B along with control miRNA or miR-487b-3p.
Data are represented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.
See also Figure S6.

Cell Reports 16, 2456–2471, August 30, 2016 2463

Figure 5. Mex-3B Inhibits the Interaction of the miR-487b-3p-Ago2 Complex with Its miR-487b-3p Target Site in the IL-33 30 UTR(A) Immunoblot analysis of Ago2 protein co-precipitated with the in vitro transcribed biotin-labeled 30 UTR-9. Cell lysates mixed at 1:1 ratio as indicated were usedin this assay. (left) Input. (right) Ago2 pulled down with biotin-labeled RNA.
(B) Schematic representation of wild-type and mutated 30 UTR-9-Dseed reporters.
(C) Immunoblot analysis of exogenously expressed FLAG-tagged Mex-3B protein co-precipitated with the indicated in vitro transcribed biotin-labeled RNAs.
GAPDH 30 UTR was used as a negative control.

(legend continued on next page)

2464 Cell Reports 16, 2456–2471, August 30, 2016

DISCUSSION
Post-transcriptional control of mRNAs encoding pro-inflammatory molecules by RNA binding proteins is considered to be a
key determinant of many immunological responses (Kafasla
et al., 2014). In the present study, we generated Mex3b/
mice and found that the RNA binding protein Mex-3B plays anessential role in the development of allergic airway inflammation.
Mex3b/ mice had fewer inflammatory cell infiltrates and bronchovascular bundles as well as lower mucin production in pulmonary tissues compared to wild-type controls. Furthermore,Th2 cytokines such as IL-4, IL-5, and IL-13 were induced lessefficiently in Mex3b/ mice. Although the expression of Mex3B in CD4+ T cells was suppressed by stimulation with antiCD3/CD28 antibody in vitro, our bone marrow transplantation
experiments suggest that the functions of Mex-3B in hematopoietic cells are not crucial for the development of allergic airway
inflammation. Additionally, we observed that Mex-3B is upregulated in airway epithelial cells, but not in non-epithelial cells
during the development of allergic airway inflammation. We
therefore focused on inflammatory factors produced by airway
epithelial cells and found that IL-33 induction is suppressed in
Mex3b/ mice. Furthermore, we showed that Mex-3B directly
binds to the 30 UTR of IL-33 and post-transcriptionally activates
its expression. Thus, our results suggest that Mex-3B plays animportant role in the development of airway inflammation by
inducing IL-33, a cytokine required for the initiation of allergic
airway inflammation.
Consistent with this notion, the degree of airway inflammationin Mex3b/ mice provoked by intranasal administration of recombinant IL-33 was comparable to that in wild-type mice. In
particular, we note that although several studies have shownthat Th2 cytokines are post-transcriptionally regulated by RNAbinding proteins such as HuR (Casolaro et al., 2008; Yarovinsky
et al., 2006), the levels of Th2 cytokines induced by the administration of IL-33 in Mex3b/ mice were comparable to those seen
in wild-type mice. Thus, the effect of Mex-3B on Th2 cytokine
expression appears to be indirect.
It has been shown that miRNAs play important roles in thedevelopment of allergic airway inflammation (Simpson et al.,2014). For example, it has been reported that miR-155 is overexpressed in the nasal mucosa of patients with allergic rhinitis andthe skin of patients with atopic dermatitis (Suojalehto et al.,2013). It has also been shown that miR-155 deficiency abolishes
allergic airway inflammation (Malmhäll et al., 2014). In this study,
we found that miR-487b-3p directly suppresses IL-33 expression and that Mex-3B upregulates IL-33 by inhibiting the functionof miR-487b-3p. Consistent with our results, it has recently beenreported that miR-487b-3p negatively regulates macrophageactivation by targeting IL-33 production (Xiang et al., 2016).
Furthermore, we showed that inhalation of anti-miR-487b-3p

restores IL-33 expression and causes airway inflammation in
Mex3b/ mice.
We found that the expression levels of miR-487b-3p are
similar between control mice and OVA-challenged wild-type
and Mex3b/ mice. Thus, the suppression of IL-33 expression
observed in OVA-challenged mice may be not be the simple
result of altered expression of miR-487b-3p. Since the expression of Mex-3B is also not altered, activation of Mex-3B, presumably by modification and/or association with other proteins,
may play an important role in the upregulation of IL-33 in
OVA-challenged mice. Consistent with our results, it has
recently been reported that miRNA-mediated mRNA repressionin vivo does not necessarily require an alteration in specificmiRNA expression (La Rocca et al., 2015) but may also be
caused by alterations in the RISC assembly in response to intracellular signaling. Thus, it is possible that upregulation of IL-33
is induced by alterations in RISC assembly in OVA-challenged
wild-type mice.
We showed that Mex-3B inhibits the interaction of the miR487b-3p-Ago2 complex with the miR-487b-3p target site in the
IL-33 30 UTR. However, this inhibition may be indirect, since
we found that the Mex-3B- and miR-487b-3p binding sites do
not overlap. We therefore speculate that Mex-3B may induce achange in the IL-33 30 UTR secondary structure that inhibits access of the miR-487b-3p-RISC complex. In line with this notion,it has been reported that the RNA binding protein Pumilio 1(Pum 1) binds to the 30 UTR of the p27 mRNA and induces achange in its secondary structure, which exposes miRNA target
sites and promotes miRNA-mediated translational repression(Kedde et al., 2010). In addition, we found that Mex-3B binds
only weakly to 30 UTR-9-DSL, a mutant lacking a predicted
stem-loop structure close to the miR-487b-3p binding site in
the IL-33 30 UTR. However, this result does not tell us whether
the stem-loop structure itself is important for Mex-3B binding:
it is also possible that this deletion simply removes a conventional binding sequence for Mex-3B. It is further possible that
deletion of the stem-loop structure induces a change in mRNA
secondary structure that is not favorable for Mex-3B binding.
We also observed that the miR-487b-3p-RISC complex binds
poorly to 30 UTR-9-DSL, which could be similarly explained bya change in mRNA secondary structure. Further study is needed
to determine the detailed regulatory mechanisms underlying
Mex-3B-mediated upregulation of IL-33.
It has recently been reported that the expression levels of
miRNAs can be regulated by their target mRNAs (Ameres et al.,2010; de la Mata et al., 2015). These reports show that target
mRNAs accelerate the decay rate of miRNAs. Consistent withthese reports, we found that knockdown of Mex-3B results ina decrease in miR-487b-3p expression. Our results suggest
that, in the absence of Mex-3B, miR-487b-3p targets IL-33,
and IL-33 may, in turn, accelerate the decay rate of miR-487b-3p.

(D) Predicted secondary structure of 30 UTR-9. A characteristic stem-loop structure is enlarged in the right panel. The miR-487b-3p target site is also indicated.
(E) Luciferase activity of A549 cells transfected with the indicated reporter plasmid along with control vector or FLAG-tagged Mex-3B.
(F) Immunoblot analysis of exogenously expressed FLAG-tagged Mex-3B protein co-precipitated with the indicated in vitro transcribed biotin-labeled RNAs.
GAPDH 30 UTR was used as a negative control.
(G) Immunoblot analysis of Ago2 co-precipitated with the indicated in vitro transcribed biotin-labeled RNAs. Cell lysates mixed at 1:1 ratio as indicated were usedin this assay. Data are represented as mean ± SD (n = 3).

Cell Reports 16, 2456–2471, August 30, 2016 2465

Figure 6. miR-487b-3p Is Important for the Development of Allergic Airway Inflammation(A) Protocol for the administration of anti-miR-487b-3p into OVA-sensitized Mex3b+/+ and Mex3b/ mice.
(B) Bronchoalveolar lavage (BAL) fluid analyses. Leukocytes were identified by morphologic criteria.
(C) Immunoblot analysis of IL-33 protein in lungs. Data are represented as mean ± SEM (n = 3–7). *p < 0.05, **p < 0.01.
See also Figure S7.

Antisense oligonucleotides have been shown to be usefulfor the treatment of various diseases, including cancer andviral, inflammatory, and neurodegenerative diseases (Evers
et al., 2015; Lindow and Kauppinen, 2012). For instance, simultaneous administration of two modified phosphorothioate antisense oligonucleotides targeting CCR3 and CCR4 (ASM8) has
been shown to efficiently inhibit allergen-induced eosinophilia

2466 Cell Reports 16, 2456–2471, August 30, 2016

and airway dysfunction in sensitized rats and human asthmatics(Allakhverdi et al., 2006). These reagents are currently being
evaluated in phase II clinical trials of asthma (Pharmaxis,2012). We have also found that nebulized administrationof an antisense oligonucleotide targeting Mex-3B (LNA-antiMex-3B) suppresses the development of allergic airway
inflammation. In our experiments, we used LNA antisense(legend on next page)

Cell Reports 16, 2456–2471, August 30, 2016 2467

oligonucleotides, which contain an engineered O20 to C40 linkage within the ribose sugar and have markedly high target
mRNA binding affinity and tissue stability (Lundin et al., 2015).
We speculate that LNA-anti-Mex-3B could hold promise asa novel anti-asthma reagent. In particular, LNA-anti-Mex-3B
could be effective against severe steroid-resistant asthma, adisease in which IL-33 plays an important role by promoting
airway remodeling (Saglani et al., 2013).
Accumulating evidence indicates that IL-33 also plays criticalroles in the development of various other inflammatory diseases,
including chronic obstructive pulmonary disease (COPD) (Byers
et al., 2013), inflammatory bowel diseases (Nunes et al., 2014),
and atopic dermatitis (Kim and Artis, 2015). It would therefore
be interesting to examine whether Mex-3B is involved in these inflammatory diseases and whether LNA-anti-Mex-3B is effective
against them.
In conclusion, we showed that Mex-3B-mediated upregulation of IL-33 is critical for the development of allergic airway
inflammation. We further showed that Mex-3B upregulates IL33 by inhibiting miR-487b-3p-mediated repression of IL-33.
Our results suggest that LNA-anti-Mex-3B could be useful forthe treatment of inflammatory diseases in which the Mex-3BIL-33 axis plays critical roles.
EXPERIMENTAL PROCEDURES
Mex3b/ MiceTo construct the Mex-3B targeting vector, exon 1-2 of the Mex3b gene was replaced with a neomycin-resistant gene cassette using the bacterial artificial
chromosome system (Yang and Seed, 2003). BALB/c embryonic stem cells
were electroporated with the linearized Mex-3B targeting vector. G418-resistant clones were screened for homologous recombination by fluorescentin situ hybridization and PCR. Mex3b+/ mice were intercrossed to generate
Mex3b/ mice. All mice were housed under specific pathogen-free conditions
at the Institute of Molecular and Cellular Biosciences, and animal care andexperimental procedures were performed in accordance with the guidelines
established by the Office for Life Science Research Ethics and Safety of the
University of Tokyo.
In Vivo Treatment of Mice
For the OVA-induced model of allergic airway inflammation, mice were
immunized as reported previously (Nakagome et al., 2009). For treatment
with LNA-anti-miR-487b-3p, mice were exposed to LNA-control or LNAanti-miR-487b-3p in PBS for 20 min on days 17–20. For treatment with
LNA-anti-Mex-3B, mice were exposed to LNA-control or LNA-anti-Mex-3B
in PBS for 20 min on days 12–17. Twenty-four hours after the final treatment,
samples of BAL fluid and lungs were obtained for examination of the BAL cell
profiles and lung histology. After centrifugation, BAL cells were resuspended
in PBS and counted with a hemocytometer. A portion of each right lung
was lysed in Trisure (Bioline) for qRT-PCR analysis or in RIPA-T buffer(50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 0.5% NaDOC,
0.1% SDS) for immunoblotting, respectively. The left lungs were homogenized in 1.0 ml of PBS containing 0.5% Triton X-100 and complete proteaseinhibitor cocktail (Roche) and used for measurements of cytokine concentrations. Bronchial lymph nodes were also examined. Recombinant IL-33 (Invi-

trogen) treatment was performed as described previously (Kondo et al.,2008). Then, 24 hr after the final treatment, BAL fluid and lung tissues were
harvested.
Histology
Tissues were fixed in 10% neutralized buffered formalin (Nacalai Tesque) andembedded in paraffin. Sections were subjected to H&E or periodic acid-Schiffstaining. For immunohistochemical staining of lung tissues, rehydrated antigen-retrieved sections were incubated with anti-IL-33 antibody (AF3636:
R&D Systems) and visualized by the avidin-biotin complex method using thechromogen diaminobenzidine (Vector Laboratories). For quantification of IL33-positive cells, a total of 400 alveolar epithelial cells were counted and cells
staining more intensely than the threshold value were counted as IL-33 positive. Results were expressed as the percentage of IL-33-positive cells over
the total number of lung epithelial cells. Quantification was performed using
automated computerized image analysis (CellSens dimension: Olympus).
For immunofluorescence staining, nuclei were stained with DAPI (40 6-diamidino-2-phenylindole; Molecular Probes) and observed by fluorescence microscopy (BZ-X700; KEYENCE).
Cell Culture and Transfection
A549 cells were cultured in complete DMEM (Nissui) supplemented
with 10% fetal bovine serum (FBS). MLE-15 cells were cultured in RPMI1640 medium (Sigma-Aldrich) supplemented with 5% FBS, ITS (5 mg/ml
insulin, 5 mg/ml transferrin, 5 mg/ml selenium: Sigma-Aldrich), 10 nM hydrocortisone (Sigma-Aldrich), 10 nM b-estradiol (Sigma-Aldrich), and 10 mM
HEPES (GIBCO). Spleen cells were cultured in complete DMEM supplemented with 10% FBS. Alveolar macrophage, bronchial lymph node cells
and CD4+ T cells were cultured in RPMI-1640 medium supplemented
with 10% FBS. Plasmids were transfected into A549 or MLE-15 cellswith Lipofectamine LTX (Invitrogen) or Lipofectamine 2000 (Invitrogen),respectively. Small oligonucleotides (siRNAs, miRNAs, and LNA oligonucleotides) were transfected into cells with Lipofectamine RNAiMAX(Invitrogen).
Immunoblotting
Cells were lysed in RIPA-T buffer. Samples were separated by SDS-PAGE
and probed with specific antibodies. Antibodies were diluted as follows:
anti-Mex-3B antibody (Ab) (Atlas), 1:1,000; anti-FLAG M2 Ab (Sigma-Aldrich),
1:2,000; anti-a-tubulin Ab (Calbiochem), 1:2,000; anti-IL-33 Ab (AF3626:
R&D Systems), 1:1,000; anti-Ago2 Ab (Wako Pure Chemicals Industries),
1:500. a-tubulin was used as a loading control.
Oligonucleotide
Stealth siRNA targeting Mex-3B and Negative Control siRNA were purchasedfrom Invitrogen. The sequences of siRNA targeting the mouse Mex3b cDNA
were CACTCAGCTTTGCTCACAATGGGAA for siRNA-1 and TATGGTTTGTAATCCGTCACAATCG for siRNA-2. Synthetic miRNAs and mirVana mimic negative control were purchased from Ambion. The LNA oligonucleotides weresynthesized as unconjugated and fully phosphorothiolated oligonucleotides(Gene Design). The perfectly matching LNA-anti-miR-487b-3p oligonucleotide:
50 -AtgamCmCcTgTamCGatT-30 (uppercase, LNA; lowercase, DNA) was complementary to nucleotides 1–16 in the mature miR-487b-3p sequence. The
mismatch control LNA oligonucleotide (LNA-anti-miR-control) was synthesizedwith the following sequence: 50 -aTgamCTcTaTaTGacT-30 . For immunofluorescence analysis, Alexa 488 was conjugated to the 50 UTR of LNA-anti-miR487b-3p. LNA-anti-miR-control or LNA-anti-Mex-3B were synthesized by
Exiqon.

Figure 7. Inhalation of Antisense Oligonucleotides Targeting Mex-3B Suppresses Allergic Airway Inflammation(A) Effect of LNA-anti-Mex-3B on Mex-3B and IL-33 protein in MLE-15 cells.
(B) Protocol for the administration of LNA-anti-Mex-3B into OVA-sensitized wild-type mice.
(C) Bronchoalveolar lavage (BAL) fluid analyses. Leukocytes were identified by morphological criteria.
(D) Lung sections were stained with anti-IL-33 antibody and counterstained with hematoxylin. The numbers of IL-33-positive cells were counted. Scale bar,
20 mm. Data are represented as mean ± SEM (n = 3–7). *p < 0.05, **p < 0.01.

2468 Cell Reports 16, 2456–2471, August 30, 2016

RNA Immunoprecipitation Assay
MLE-15 cells were lysed in NP-40 buffer (20 mM Tris-HCl [pH 7.5], 150 mM
NaCl, 1 mM EDTA, 0.5 mM DTT, 0.5% Nonidet P-40, 120 U/ml RNase inhibitor,
protease inhibitor cocktail). A549 cells were transfected with the Mex-3B
expression constructs. Twenty-four hours after transfection, cytoplasmic cell
extracts were prepared in buffer B (10 mM PIPES [pH 6.8], 300 mM sucrose,
100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 120 U/ml RNase
inhibitor, protease inhibitor cocktail). Immunoprecipitation was performed using Dynabeads protein G (Invitrogen). The beads were washed and bound RNA
was eluted from the beads by directly adding Trisure (Bioline), followed by RNA
extraction and qRT-PCR.
In Vitro Transcription
Murine IL-33 30 UTRs were cloned into pBluescript II SK (+) at the KpnIand XhoI sites. These constructs were linearized by digestion with EcoRIand in vitro transcription was performed with the T7 MAXIscript Kit(Ambion). Biotin-labeled RNAs were synthesized in the presence of biotinlabeled dCTP during in vitro transcription. GAPDH 30 UTR was used as acontrol.

ACKNOWLEDGMENTS
We are grateful to R. Le Goffic for A549 and MLE-15 cells. We thank K. Watanabe, M. Kobayashi, and A. Miyazawa for genotyping, A. Kamiya and H. Matsuoka for discussion, help, and support, and A. Kawai and Y. Koreeda for technical assistance in generating knockout mouse lines. This work was supported
by Grants-in-Aid for Scientific Research on Innovative Areas (Integrative
Research on Cancer Microenvironment Network) and Research Program of
the Project for Development of Innovative Research on Cancer Therapeutics(P-Direct), Research Program of Innovative Cell Biology by Innovative Technology (Integrated Systems Analysis of Cellular Oncogenic Signaling Networks), Ministry of Education, Culture, Sports, Science and Technology ofJapan.
Received: October 20, 2015
Revised: June 20, 2016
Accepted: July 25, 2016
Published: August 18, 2016

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mRNA Binding Protein Pull-down Assay
A549 cells were transfected with the Mex-3B expression construct. Twentyfour hours after transfection, cells were lysed in IP buffer (10 mM Tris-HCl[pH 7.5], 100 mM NaCl, 2.5 mM MgCl2, 0.5% Triton X-100, 120 U/ml RNase
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Statistics
Statistical analysis was performed by unpaired Student’s t test. Differenceswere considered statistically significant when p < 0.05.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and one table and can be found with this article online at
http://dx.doi.org/10.1016/j.celrep.2016.07.062.
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