Interleukin-1-inducible MCPIP protein has structural and
functional properties of RNase and participates in
degradation of IL-1b mRNA
Danuta Mizgalska
1
, Paulina We˛ grzyn
1
, Krzysztof Murzyn
2
, Aneta Kasza
1
, Aleksander Koj
1
,
Jacek Jura
3
, Barbara Jarza˛b
4
and Jolanta Jura
1
1 Department of Cell Biochemistry, Jagiellonian University, Krakow, Poland
2 Department of Biophysics, Jagiellonian University, Krakow, Poland
3 National Research Institute of Animal Production, Balice, Poland
4 M. Sklodowska-Curie Memorial Institute, Gliwice, Poland
Introduction
Macrophages and hepatic cells are important players
in the inflammatory processes initiated in response to a
variety of agents, including viral or bacterial infections,
thermal and mechanical trauma, or malignant growth.
Recently, we have used human monocyte-derived mac-
rophages exposed to interleukins IL-1b and IL-6, and

ning amino acids 130-280; such domains are known to possess structural
features of RNases. Recently, RNase properties of MCPIP were confirmed
on transcripts coding for interleukins IL-6 and IL-12p40. Here we present
evidence that siRNA-mediated inhibition of the MCPIP gene expression
increases the level of the IL-1b transcript in cells stimulated with LPS,
whereas overexpression of MCPIP exerts opposite effects. Cells with an
increased level of wild-type MCPIP showed lower levels of IL-1b mRNA.
However, this was not observed when mutant forms of MCPIP, either
entirely lacking the PIN domain or with point mutations in this domain,
were used. The results of experiments with actinomycin D indicate that lower
levels of IL-1b mRNA are due to shortening of the IL-1b transcript half-life,
and are not related to the presence of AU-rich elements in the 3¢ UTR. The
interaction of the MCPIP with transcripts of both IL-1b and MCPIP
observed in an RNA immunoprecipitation assay suggests that this novel
RNase may be involved in the regulation of expression of several genes.
Abbreviations
ARE, AU-rich element; dsRBD, double-stranded RNA-binding domain; GM-CSF, granulocyte ⁄ macrophage colony-stimulating factor; iNOS,
inducible nitric oxide synthase; IL-1b, interleukin-1b; IL-6, interleukin-6; KH domain, K homology domain; LPS, lipopolysaccharide; MCP-1,
monocyte chemoattractant protein-1; MCPIP, MCP-1 induced protein; PAZ, Piwi Argonaut and Zwille; PDB, protein data bank; PMA, phorbol
12-myristate-13-acetate; RRM, RNA-recognition motif; TNF, tumor necrosis factor.
7386 FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS
finger motif (CCCH) in MCPIP, which prompted
Zhou et al. to propose a hypothetical function for this
molecule as a novel transcription factor [2]. They also
showed that MCPIP is a negative regulator of macro-
phage activation, affecting LPS-induced tumor necrosis
factor (TNF) and inducible nitric oxide synthase
(iNOS) promoters [3].
Recently, Matsushita et al. [4] reported that MCPIP
has an essential role in preventing immune disorders.

was confirmed by an RNA immunoprecipitation assay.
Our findings are in agreement with those of Mats-
ushita et al. [4]. The data show that MCPIP has more
inflammatory targets than described so far, and that
this protein is an important regulator of inflammatory
processes.
Results
Influence of proinflammatory cytokines on MCPIP
transcript level
Real-time PCR was used to determine the modulation
of expression of the MCPIP gene in two types of cells:
HepG2 cells stimulated with IL-1b or IL-6, and mono-
cyte lymphoma cells stimulated with IL-1b, TNFa,
PMA or LPS. As shown in Fig. 1A, the MCPIP tran-
script level in HepG2 cells stimulated with IL-1b for
A
B
C
Fig. 1. Regulation of MCPIP gene expression. Changes in MCPIP
gene expression in HepG2 and U937 cells measured by real-time
PCR. The results are means ± SD of three independent experi-
ments (*P < 0.05; **P < 0.01 versus control). (A) Changes in the
expression of the MCPIP gene after stimulation of HepG2 cells
with IL-1b (15 ngÆmL
)1
). Cells were stimulated for 0.25, 0.5, 1, 2,
4, 8, 12 and 24 h. Unstimulated cells collected at each time point
served as controls. (B) Expression of the MCPIP gene after stimula-
tion of HepG2 cells with IL-6 (15 ngÆmL
)1

region of the MCPIP1 sequence [5]. Ten top scoring
Protein Data Bank (PDB) records included 1O4W
(the PIN domain from Archaeoglobus fulgidus AF0591
protein; six hits from the various fold recognition serv-
ers), 1A76 (FLAP endonuclease-1 from Methanococ-
cus jannaschii) and 1W8I (AF1683 protein of unknown
function from Archeoglobus Fulgidus). For greater con-
fidence that the structural alignments created by the
GeneSilico.pl server [6] are meaningful, we compared
the reported AF0591 PIN ⁄ MCPIP PIN alignment with
previously published alignments of human SMG6
(PDB ID: 2HWW) PIN and AF0591 PIN sequences
[7,8]. The resulting alignment is shown in Fig. 2B.
Four acidic residues (D141, E185, D226, D244) in PIN
domains involved in binding Mg
2+
ions are conserved
in all three sequences. Other conserved residues are
R263, V139 and N144. Determination of the MCPIP
domain architecture was completed using the DISO-
PRED [9] and SPRITZ servers [10], which consistently
indicated that regions 1-50, 90-130 and 290-540 of
MCPIP are disordered, with as yet unidentified func-
tion (Fig. 2A).
Distribution of MCPIP transcript in human tissues
and cellular localization of MCPIP protein
A blot loaded with mRNAs from several human tis-
sues was subjected to Northern blot analysis using a
molecular probe spanning the entire coding sequence
of the MCPIP gene or the b-actin gene (control). In

ized in the cytoskeleton fraction (Fig. 3B). In the
second approach, HepG2 cells were transfected either
with an empty vector, or with a vector expressing a
fusion of MCPIP with red fluorescent protein (RFP).
Using various concentrations of the plasmid vector
overexpressing the fusion protein MCPIP–RFP, we
observed that the protein was localized in the cyto-
plasm, in the form of granules (Fig. 3C). It is possible
that formation of granules is the result of stress (e.g.
related to overexpression). Such accumulation of pro-
tein has been described previously for other factors
engaged in mRNA degradation: for example, triste-
traproline and T-cell-restricted intracellular antigen 1
both accumulate in stress granules during environmen-
tal stress, which are regarded as dynamic cytoplasmic
foci that contain untranslated mRNAs [11,12].
Furthermore, the results of western blots show that
MCPIP is detected in cytoskeleton. These data are in
agreement with results obtained by Henics et al. [13],
who showed that cytoskeleton proteins have binding
capacity for proteins involved in mRNA metabolism.
Moreover, cytoskeleton proteins, such as actin or
tubulin, are thought to be crucial for compartmentali-
zation and translation of various mRNAs, including
those containing AU-rich instability motifs in the 3 ¢
UTR [14,15]. We performed co-immunoprecipitation
and mass spectrometry analyses to identify putative
proteins interacting with MCPIP (data not shown). Of
the identified proteins, 73% are proteins involved in
mRNA stability, but there are also proteins involved

Membrane
Nucleus
Cytoskeleton
Liver
Small intestine
Placenta
Lungs
Leukocytes
Fig. 3. Tissue distribution of transcript and cellular localization of
MCPIP. (A) A human tissue Northern blot (Clontech) was hybridized
with a radioactively labelled MCPIP cDNA probe and visualized by
autoradiography. A b-actin probe served as a control for equal loading.
A single 2.0 kb band was seen in all lanes. A 1.8 kb actin isoform is vis-
ible for heart and skeletal muscle. The duration of exposure of the
Northern blot with MCPIP probe was 20 times longer compared to the
blot with the b-actin probe. (B) A Qproteome kit (Qiagen) was used to
isolate cytosolic, membrane, nuclear and cytoskeletal proteins. Pro-
tein concentrations were determined using a BCA protein assay
(Sigma). Aliquots of 10 lg from each fraction were used in western
blot analysis. Polyclonal antibodies against MCPIP were used at
concentration of 1 lgÆmL
)1
. As markers specific for cytoskeleton,
nucleus, membrane and cytosolic fraction, respectively, antibodies
against actin (mouse monoclonal, 1:4000, Sigma), lamin C (rabbit poly-
clonal, 1:1000, Abcam), MnSOD (rabbit polyclonal, 1:2000, Sigma)
and GAPDH (rabbit polyclonal, 1:1000, Abcam) were used. (C) A
genetic construct overexpressing recombinant MCPIP with fluores-
cent red protein in the pmaxFP-Red-N vector (Amaxa) was generated
for determination of the cellular localization of MCPIP. HepG2 cells

MCPIP in comparison to cells transfected with an
empty vector or one expressing D ⁄ A-MCPIP (Fig. 5,
lanes 2,3 and 4, lower band). The levels of mutant and
wild-type MCPIP mRNAs were higher when these
vectors were co-transfected with a vector overexpress-
ing IL-1b (Fig. 5, lanes 3 and 4, upper bands). The
absence of a plasmid encoding IL-1b prevented the
increase in the MCPIP mRNA level for both forms
(Fig. 5A, lanes 5 and 6, upper bands). Moreover, in a
co-transfection study with the IL-1b-expressing vector,
the level of mRNA for the mutant form of MCPIP was
higher (Fig. 5A, lane 4, upper band) than when wild-
type MCPIP was co-transfected (Fig. 5A, lane 3, upper
band). At this stage of the study, the following hypo-
thesis may be presented: some overexpressed IL-1b is
secreted and stimulates the endogenous form of MCPIP,
resulting in the observed higher level of MCPIP when
A
B
Fig. 4. MCPIP regulates IL-1b transcript in LPS-treated human
fibroblasts. (A) Human fibroblasts were transfected with siRNA
specific either for MCPIP (50 n
M)orGAPDH (50 nM), or with non-
specific siRNA (negative control, 50 n
M). The transfection efficiency
observed for carboxyfluorescein (FAM)-labeled scrambled siRNA
was estimated as 25%. Cells treated only with the transfection
reagent siPORT NeoFX were used as a control. At 48 h post-trans-
fection, cells were treated with LPS (100 ngÆmL
)1

HepG2 cells overexpressing IL-1b and its absence in
cells transfected with an empty vector (data not shown).
Wild-type MCPIP (endogenous and exogenous) starts
degradation of IL-1b mRNA and also of its own tran-
script. For this reason, the level of mRNA for the wild-
type form of MCPIP and the level of mRNA for IL-1b
(Fig. 5A, lane 3) were lower in comparison to the level
of mRNA for MCPIP with a mutation within the PIN
domain (Fig. 5A, lane 4). The same tendency is seen at
the protein level (data not shown).
In order to determine whether the observed IL-1b
mRNA changes are due to a decrease in transcription
or an increased degradation rate, we performed experi-
ments with actinomycin D. HepG2 cells stably
overexpressing IL-1b mRNA were co-transfected with
an empty vector or a vector overexpressing wild-type
MCPIP or its mutant forms [mutMCPIP (without the
PIN domain) and D ⁄ A-MCPIP (with two mutated
residues within PIN domain)]. The cells were harvested
0, 3, 6 and 9 h after addition of actinomycin D, and
the level of exogenous IL-1b mRNA was analyzed by
Northern blot. A significant reduction in the IL-1b
transcript level was observed only when the wild-type
MCPIP was overexpressed (Fig. 5B).
Importance of AREs in regulation of IL-1b mRNA
by MCPIP
In order to determine whether AREs are important in
mRNA degradation triggered by MCPIP, we used
A
B

independent experiments. The panel on the right shows densitometric measurements of the IL-1b transcript level normalized to the 18S
rRNA band. Student’s t test was used to determine significant differences from the control (*P < 0.05, **P < 0.01). (C) Two genetic con-
structs consisting of a minimal promoter and cDNA for luciferase with (p-luc-ARE) or without AREs (p-luc) from the IL-1b transcript were
generated. HepG2 cells were co-transfected with combination of p-luc-ARE and pcDNA3 empty vector (control), or wild-type MCPIP or a
mutant form of MCPIP lacking the domain with RNase activity. The same combinations were used for the plasmid expressing luciferase
transcript without ARE sequences in the 3¢ UTR (p-luc). As an internal transfection control, a pEF1 ⁄ Myc-His ⁄ Gal vector encoding b-galactosi-
dase was used. The bars represent the luciferase activity calculated from three independent experiments, normalized to that for b-galactosi-
dase. Overexpression of MCPIP was confirmed by western blotting (right upper panel). Semi-quantitative RT-PCR was performed for the
luciferase transcript to show the reporter transcript level (lower panel). The GAPDH
transcript served as a housekeeping gene.
MCPIP protein as an RNase D. Mizgalska et al.
7392 FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS
three genetic constructs: (a) a construct overexpressing
IL-1b transcript with AREs (long 3¢ UTR, Fig. 6A),
(b) a construct overexpressing IL-1b transcript without
AREs (short 3¢ UTR, Fig. 6A), and (c) a construct
overexpressing the MCPIP transcript. HepG2 cells
were co-transfected with vector containing IL-1b
cDNA with or without AREs or with the vector con-
taining MCPIP cDNA. The levels of IL-1b transcript
with ⁄ without AREs and the level of MCPIP transcript
were determined using Northern blotting 24 h after
transfection. The endogenous MCPIP transcript is not
detectable due to the short exposure time.
The level of IL-1b transcripts containing AREs was
lower than the level of IL-1b mRNA without AREs
(Fig. 6B, lanes 2–5). These results are in agreement with
data indicating that transcripts with AREs are more sus-
ceptible to degradation [16]. In both cases, overexpres-
sion of MCPIP (IL-1b + ARE and IL - 1b )ARE)

the control and samples overexpressing wild-type
MCPIP or its mutant form lacking the PIN domain.
Nevertheless, the levels of luciferase transcript
(Fig. 6C, right panel) and luciferase activity (Fig. 6C,
left panel) were significantly lower in all samples
(including control) when AREs were present. This
A
B
Fig. 7. RNA immunoprecipitation. (A) Control of the experimental model. HepG2 cells were transfected with a vector overexpressing wild-
type MCPIP. RNA was isolated and cDNA synthesis was performed. Expression of genes encoding exogenous MCPIP (cells with a vector
overexpressing MCPIP) or endogenous transcripts encoding IL-1b and GADPH was determined by semi-quantitative PCR (with EF-2 as a
reference transcript). A total RNA sample (0.1 lg RNA) served as a negative control (cDNA-). (B) To bind RNA templates to proteins,
HepG2 cells were subjected to crosslinking with formaldehyde, lysed and incubated with antibodies specific for MCPIP or with IgG as a
negative control. Then the samples were digested with DNase to remove traces of genomic DNA. Reverse transcription was performed
using RNA isolated from immunoprecipitates, followed by PCR with MCPIP-, IL-1 b- and GADPH-specific primers (35 cycles). The same RNA
but not reverse-transcribed served as a negative control for the PCR reaction. Specific transcripts coding for MCPIP and IL-1b were detected
only in immunoprecipitates obtained with antibodies specific for MCPIP, but not with IgG antibodies. Specificity of the PCR product was
determined by restriction analysis (data not shown). PCR product was not observed for the negative control, i.e. with primers specific for
the GAPDH gene.
D. Mizgalska et al. MCPIP protein as an RNase
FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS 7393
means that IL-1b AREs are important in luciferase
mRNA degradation but this process is performed in
an MCPIP-independent manner.
Interaction of MCPIP protein with the IL-1b
transcript
An RT-PCR reaction was performed to confirm that
HepG2 cells synthesize endogenous IL-1b mRNA. As
shown in Fig. 7A, there is expression of the gene
encoding IL-1b, although the transcript level is low in

ferons b and c) contain regulatory elements in the 3¢
UTR that are important for the half life of cytokine
mRNAs. For this reason, transcription of genes encod-
ing cytokines is not sufficient to guarantee the appear-
ance of functional proteins. As reported by Kaspar
and Gehrke [18] and Dinarello [19], stimulation of
macrophages by mild stimulants (such as C5a comple-
ment or some modified proteins) leads to accumulation
of specific mRNAs without their translation. This is
due to the formation of translationally inactive
mRNA–protein complexes. These complexes may
either result in mRNA degradation or its conversion
into translationally active forms by the action of
strong stimulants, such as endotoxin (LPS).
One of the regulatory sequences involved in deter-
mining mRNA half life are the AREs, which can stim-
ulate deadenylation of mRNA [20] and its degradation
by the exosome in the 3¢fi5¢ direction [21]. In addi-
tion to mRNA degradation mediated by the exosome,
RNA can be degraded through removal of the
7-methyl guanosine cap, followed by 5¢fi3¢ mRNA
degradation in cytoplasmic processing bodies
(P-bodies) [22]. One of the best known examples of
AREs binding proteins involved in inflammatory
processes is tristetraprolin [23]. It was shown that
tristetraprolin enhances the deadenylation and
degradation of mRNA for granulocyte ⁄ macrophage
colony-stimulating factor, TNFa and IL-2.
Using human monocyte-derived macrophages
exposed to IL-1b, we found that the MCPIP gene is

mRNA degradation, with IL-1b mRNA being one of
the targets. To confirm our hypothesis, we used genetic
constructs overexpressing full-length cDNA for IL-1b,
wild-type MCPIP and its mutant forms. The level of
IL-1b mRNA was decreased in cells overexpressing
MCPIP protein as an RNase D. Mizgalska et al.
7394 FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS
wild-type MCPIP in comparison with a mutant form.
Inhibition of MCPIP expression by siRNA leads to
the increase in IL-1b transcript level. These data are in
agreement with the results obtained by Liang et al. [3].
MCPIP-mediated downregulation of the half life of the
IL-1b transcript was confirmed in the presence of acti-
nomycin D in HepG2 cells stably overexpressing IL-1b
mRNA. We found that the stability of IL-1b transcript
is dependent on the type of MCPIP expressed by the
vector introduced into these cells. In control cells
(empty vector) and cells overexpressing a mutant form
of MCPIP (without the PIN domain, or MCPIP with
two mutated amino acid residues), the transcript cod-
ing for IL-1b was stable. However, when wild-type
MCPIP was present, a decrease in the transcript level
for IL-1b was observed. We have also shown that
IL-1b transcript degradation is ARE-independent. We
used genetic constructs with cDNA coding for interleu-
kin-1 but either containing or lacking the region with
ARE sequences. Co-transfection of constructs with or
without ARE sequences with the vector overexpressing
MCPIP resulted in efficient disappearance of the IL-1b
transcript. The degradation was independent of the

Our results confirm the data of Matsushita et al. [4]
showing that MCPIP has RNase properties performed
by the PIN domain. We found that MCPIP is acti-
vated by many stimulants, and regulates the level of
IL-1b mRNA and of its own transcript. Further stud-
ies are necessary to clarify the biological role of
MCPIP as a nuclease and to identify the cis-acting
sequences in the 3¢ UTR of the IL-1b and MCPIP
transcripts that directly interact with MCPIP. How-
ever, as MCPIP regulates the stability of transcripts
encoding the two major proinflammatory cytokines
IL-1b and IL-6, as well as of its own transcript, it may
be regarded as a very important potential regulator of
inflammatory processes.
Experimental procedures
Cell culture and stimulation with cytokines
All cell lines were grown under standard conditions ()37 °C
and 5% CO
2
). HepG2 cells (American Type Culture Collec-
tion, Manassas, VA, USA) were cultured in Dulbecco’s mod-
ified Eagle’s medium with 1000 mgÆL
)1
d-glucose (Gibco,
Carlsbad, CA, USA) supplemented with 5% fetal bovine
serum. Cells were grown in plastic Petri dishes (6 cm in diam-
eter, BD Biosciences, Franklin Lakes, NJ, USA) to approxi-
mately 70% confluence. At 15 h before stimulation with
cytokine, cells were washed twice in NaCl ⁄ P
i

performed using SYBR Green PCR master mix (DyNAmoÔ
HS SYBR Green qPCR; Finnzymes, Espoo, Finland), 1 ll
cDNA and 20 ng suitable forward and reverse primers. For
D. Mizgalska et al. MCPIP protein as an RNase
FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS 7395
amplification, the following primers were used: IL-1b cDNA,
5¢-GATGTCTGGTCCATATGAACTG-3¢ (forward) and
5¢-TGGGATCTACACTCTCCAGC-3¢ (reverse); MCPIP
cDNA, 5¢-GGAAGCAGCCGTGTCCCTATG-3¢ (forward)
and 5¢-TCCAGGCTGCACTGCTCACTC-3¢ (reverse). Each
sample was normalized to reference genes: elongation
factor 2 (EF-2), 5¢-GACATCACCAAGGGTGTGCA-3¢
(forward) and 5¢-TTCAGCACACTGGCATAGAGGC-3¢
(reverse); GAPDH,5¢-CCGAGCCACATCGCTCAGAC-3¢
(forward) and 5¢-GTTGAGGTCAATGAAGGGGTC-3¢
(reverse). The relative level of transcripts was quantified by
the DDC
T
method.
Northern blot analysis
For determination of MCPIP and IL-1b transcript levels,
10 lg of total RNA were separated in a 1% formaldehyde
agarose gel and blotted to a nitrocellulose membrane. Pre-
hybridization and hybridization were performed at 65 °Cin
1% SDS, 1 m NaCl, 10% dextran sulfate solution. Probes
were generated by PCR amplification of the entire coding
region of studied genes. A random primer labelling kit
(Promega) was used to label 30 ng of PCR product with
[a-
32

GCGGGTAGG-3¢ and reverse primer 5¢-GGGGTGGGCC
TCAGGGCTGGG-3¢. Then nested primers 5¢-GTCTGA
GCTATGAGTGGCCC-3¢ (forward) with a restriction site
for KpnI and 5¢-CAGCTTACTCACTGGGGTGC-3¢
(reverse) with a restriction site for EcoRI were used to
obtain a region of 1813 bp, corresponding to positions -9
to 1804 relative to the start codon (ATG) of the sequence
NM_025079. The obtained fragment was cloned into a
pcDNA3 vector (Invitrogen, Carlsbad, CA).
The same sequence was used to design primers for gener-
ation of a mutant form of MCPIP, without a PIN domain
(mutMCPIP). The fragment from 412-888 bp, correspond-
ing to the PIN domain, was removed from the entire cod-
ing sequence of the MCPIP gene. The coding sequence
lacking the PIN domain was obtained in two steps. First,
the fragment of 423 bp situated in front of the PIN domain
was amplified by PCR using forward primer 5¢-
CGGGATCCCGGAGTCTGAGCTATGAGTG-3¢ with a
restriction site for BamHI and reverse primer 5¢-CACT
GGTCTCAGGTCGCTG-3¢ with a restriction site for BsaI,
and cloned into pcDNA3 at the same restriction sites. Then
a second fragment situated after the PIN domain was gen-
erated by PCR using primers 5¢-AGCGACCTGAGAC
CACTCACTTTGGAGCAC-3¢ (BsaI) and 5¢-GGAATTC
CCTCACTGGGGTGCTGG-3¢ (EcoRI). This fragment
was cloned into pcDNA3 containing the first fragment at
the BsaI ⁄ EcoRI restriction sites. A genetic construct with
mutation of two conserved amino acids residues within the
PIN domain (D141A and D226A) was generated by site-
directed mutagenesis (Stratagene, Cedar Creek, TX, USA).

IL-1b cDNA (p-luc)ARE and p-luc), the region flanking
the AREs in IL-1b cDNA was amplified using forward
primer 5¢-ATTCGCTCCCACATTCTGATG-3¢ and reverse
primer 5¢-ACACTGCTACTTCTTGCC-3¢. Both primers
had restriction sites for XbaI. The PCR product was cloned
into pGL3-Promoter (Promega) at the XbaI site, and orien-
tation of the insert was confirmed by PCR.
Transient transfection
For transient transfection experiments, HepG2 cells and
human fibroblasts were cultured in six-well plates to
approximately 80% confluency. The next day, cells were
transfected using Lipofectamine 2000 (Invitrogen) in serum-
free Opti-MEM (Invitrogen), according to the manu-
facturer’s protocol. Empty pcDNA3 vector was used as a
control for the experiment or to equalize the amount of
genetic material introduced.
siRNA transfection
The pre-designed siRNAs obtained from Ambion (Austin,
TX, USA) included GAPDH siRNA as a positive control
and siRNA with a scrambled sequence as a negative control.
We tested six different siRNA specific for the MCPIP gene.
The most potent inhibitor appeared to be the sequence 5¢-
CCCUGUUGAUACACAUUGUTT-3¢. Human fibroblasts
were cultured in 12-well plates. The siRNA concentration
and amount of transfection reagent required were optimized
experimentally. Before transfection, cells were trypsinized,
centrifuged at 1000 g at 4 °C for 5 min, and resuspended in
fresh medium. The lipid-based transfection reagent siPORT
NeoFX (4 ll per well) from Ambion and siRNAs (50 nm
final concentration) were separately diluted in 50 lL

(Tropix, Foster City, CA, USA) reporter gene as described
by the manufacturer. The luciferase activity of each con-
struct was normalized to b-galactosidase activity as an
integral transfection control. Total RNA was isolated as
described above. Semi-quantitative RT-PCR was performed
using primers specific for luciferase (forward 5¢-AGA
GATACGCCCTGGTTCCT-3¢; reverse 5¢-AATCTGACG
CAGGCAGTTCT-3¢) and GAPDH transcripts (sequences
given above).
Generation of rabbit antibodies specific for
MCPIP, and western blot analysis
E. coli BL21 cells were transfected with pQE-31 vector
(Qiagen, Du
¨
sseldorf, Germany) containing the entire coding
sequence of the MCPIP transcript. The recombinant pro-
tein was purified from bacterial culture using BD Talon
metal affinity resins with cobalt ions (BD Biosciences) and
used for immunization of a New Zealand White rabbit
(three times 300 lg of antigen). Antibodies were purified
from rabbit sera by protein A–Sepharose chromatography
and then checked for specificity. Generated antibodies were
used for western blot analysis of protein extracts from
HepG2 cells transfected with a vector overexpressing
MCPIP. Several independent experiments showed that there
was no cross-reactivity, and only a specific product of the
expected size was observed on membranes.
For subcellular fractionation of HepG2 cells overex-
pressing MCPIP, the Qproteome kit (Qiagen) was used,
and all steps were performed as described by the manufac-

Triton X-100, 1.2 mm EDTA, 16.7 mm NaCl, protease
inhibitor complex (Roche, Basel, Switzerland), 16.7 mm
Tris ⁄ HCl, pH 8]. Protein A–agarose (Roche) used for
pre-cleaning of the lysate, was washed with IP buffer,
blocked with 1 mgÆmL
)1
BSA, and 20 lgÆmL
)1
salmon
DNA (Sigma). Then the sample was divided into two parts
and antibodies were added: anti-MCPIP to the first and
rabbit polyIgG as a negative control to the second. Samples
were incubated overnight at 4 °C with constant mixing.
The next day, blocked protein A–agarose beads were
added. After 1 h of incubation at 4 °C, the immunoprecipi-
tates were centrifuged (at 14 000 g, 10 min, 4 °C) and
washed twice each with four buffers (B-I: 0.1% SDS, 1%
Triton X-100, 2 mm EDTA, 150 mm NaCl, 20 mm
Tris ⁄ HCl, pH 8; B-II: 0.1% SDS, 1% Triton X-100, 2 mm
EDTA, 500 mm NaCl, 20 mm Tris ⁄ HCl, pH 8; B-III:
0.25 m LiCl, 1% Nonidet P-40, 1% sodium deoxycholate,
1mm EDTA, 10 mm Tris ⁄ HCl, pH 8; B-IV: 1 mm
EDTA,10 mm Tris ⁄ HCl, pH 8). RNA was eluted from
beads with buffer containing 1% SDS and 0.1 m NaHCO
3
.
After addition of NaCl to 0.2 m concentration, samples
were reverse crosslinked by incubation at 65 °C for 4 h,
with subsequent protein digestion by proteinase K. RNA
was isolated from the immunoprecipitates using the phe-

Wojciechowska M, Jarza˛ b M, Kowalska M, Piechota
M, Przewocki P & Koj A (2008) Identification of inter-
leukin-1 and interleukin-6-responsive genes in human
monocyte-derived macrophages using microarrays. Bio-
chim Biophys Acta 1779, 383–389.
2 Zhou L, Azfer A, Niu J, Graham S, Choudhury M,
Adamski FM, Younce C, Binkley PF & Kolattukudy
PE (2006) Monocyte chemoattractant protein-1 induces
a novel transcription factor that causes cardiac myocyte
apoptosis and ventricular dysfunction. Circ Res 98,
1177–1185.
3 Liang J, Wang J, Azfer A, Song W, Tromp G,
Kolattukudy PE & Fu M (2008) A novel CCCH-zinc
finger protein family regulates proinflammatory activa-
tion of macrophages. J Biol Chem 283, 6337–6346.
4 Matsushita K, Takeuchi O, Standley DM, Kumagai Y,
Kawagoe T, Miyake T, Satoh T, Kato H, Tsujimura T,
Nakamura H et al. (2009) Zc3h12a is an RNase essen-
tial for controlling immune responses by regulating
mRNA decay. Nature 458, 1185–1190.
5 Wallner B & Elofsson A (2006) Identification of correct
regions in protein models using structural, alignment,
and consensus information. Protein Sci 15, 900–913.
6 Kurowski MA & Bujnicki JM (2003) GeneSilico protein
structure prediction meta-server. Nucleic Acids Res 31,
3305–3307.
7 Glavan F, Behm-Ansmant I, Izaurralde E & Conti E
(2006) Structures of the PIN domains of SMG6 and
SMG5 reveal a nuclease within the mRNA surveillance
complex. EMBO J 25, 5117–5125.

JM (2002) IL-1b transcript stability in monocytes is
linked to cytoskeletal reorganization and the availability
of mRNA degradation factors. Immunol Cell Biol 80,
328–339.
15 Sweet TJ, Boyer B, Hu W, Baker KE & Coller J (2007)
Microtubule disruption stimulates P-body formation.
RNA 13, 493–502.
16 Roca FJ, Cayuela ML, Secombes CJ, Meseguer J &
Mulero V (2007) Post-transcriptional regulation of
cytokine genes in fish: a role for conserved AU-rich
elements located in the 3¢-untranslated region of their
mRNAs. Mol Immunol 44, 472–478.
17 Paschoud S, Dgar AM, Kuntz C, Grisoni-Neupert B,
Richman L & Kuhn LC (2006) Destabilization of inter-
leukin-6 mRNA requires a putative RNA stem–loop
structure, an AU-rich element, and the RNA-binding
protein AUF1. Mol Cell Biol 26, 8228–8241.
18 Kaspar RL & Gehrke L (1994) Peripheral blood
mononuclear cells stimulated with C5a or lipopolysac-
charide to synthesize equivalent levels of IL-1beta
mRNA show unequal IL-1beta protein accumulation
but similar polyribosome profile. J Immunol 153, 277–
286.
19 Dinarello C (1996) Biologic basis for interleukin-1 in
disease. Blood 87, 2095–2147.
20 Lai WS, Carballo E, Strum JR, Kennington EA,
Phillips RS & Blackshear PJ (1999) Evidence that
tristetraprolin binds to AU-rich elements and promotes
the deadenylation and destabilization of tumor necrosis
factor alpha mRNA. Mol Cell Biol 19, 4311–4323.

FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS 7399