RHEUMATOLOGY

Rheumatology 2013;52:427437
doi:10.1093/rheumatology/kes292
Advance Access publication 12 November 2012

Original article
Intron-derived aberrant splicing of A20 transcript in
rheumatoid arthritis
Hyun Kyung Yoon1,2,*, Hee Sun Byun1,2,*, Hyunji Lee1, Juhee Jeon1,
Yoonjung Lee1, Yuwen Li1, Eun-Heui Jin2, JaeWoo Kim2, Jang Hee Hong1,2,
Jin Hyun Kim3, Jeong Ho Seok1, Seong Wook Kang3, Won Hyung Lee2 and
Gang Min Hur1,2
Abstract

Results. In RA FLSs, we discovered four novel aberrant A20 transcripts, most of which resulted from
insertion of partial intron 2, intron 4 and/or deletion of exon 4. In each of these FLSs, sequence analysis
revealed that these aberrant insertional sequences were flanked by consensus splice donor and acceptor
sequences without nucleotide substitution, suggesting alternative splicing as the likely mutational mechanism. These variants elicited a codon frame shift by creating a premature translational stop codon, and
eventually, disruption of the OTU domain (which is functionally important as an inhibitor of NF-kB activation) of A20. The expression level of aberrant A20 transcript was correlated well with persisitently
enhanced status of NF-kB signalling, as evident by the phosphorylation of inhibitor of NF-kB (IkB)-a
and transcription of NF-kB target genes.
Conclusion. The results suggest that A20 inactivation by the novel aberrant splicing may contribute to RA
progression by inducing persistent NF-kB activation.
Key words: rheumatoid arthritis, A20, aberrant splicing variants, NF-kB, tumor necrosis factor.

Introduction
RA is a common chronic inflammatory disease that affects
the small joints of the hands and feet [1]. The propagation
1
Department of Pharmacology, 2Clinical Trials Center, Research
Institute for Medical Scineces, 3Division of Rheumatology, Department
of Internal Medicine, College of Medicine, Chungnam National
University, 6 Munhwa-dong, Jung-gu, Daejeon, Korea.

Submitted 15 May 2012; revised version accepted

18 September 2012.
Correspondence to: Gang Min Hur, Department of Pharmacology,
College of Medicine, Chungnam National University, 6 Munhwa-dong,
Jung-gu, Daejeon, 301-131, Korea. E-mail: gmhur@cnu.ac.kr
*Hyun Kyung Yoon and Hee Sun Byun contributed equally to this
study.

of inflammation is characterized by migration of mononuclear cells into the local inflammatory sites, leading to the
proliferation of fibroblast-like synoviocytes (FLSs) and
damage to cartilage and bone [2]. The major mediators
of chronic inflammation in RA include TNF-a, IL-1b, IL-6,
IL-8 and PGE2 [3]. Induction of pro-inflammatory mediators requires activation of the nuclear factor (NF)-kBinducing kinase- or inhibitor of NF-kB (IkB) kinase
(IKK)-mediated NF-kB signal transduction pathways
[4, 5]. The transcription factor NF-kB has been well recognized as a key regulator of inflammation in RA [68].
NF-kB proteins are rendered inactive by binding to IkBs
in normal conditions. The activation of IKK complex by the
stimulation of multiple receptors that phosphorylate IkB at

! The Author 2012. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

BASIC
SCIENCE

Methods. Alterations in A20 transcripts were determined through sequence analysis of 10 clones of A20
cDNA in FLSs from each of the five RA patients. The levels of aberrant A20 transcript were measured by
quantitative real-time RTPCR with primers to specifically recognize the inserted introns. The functional
role of A20 and its aberrant variants were examined by analysing NF-kB luciferase reporter activity and
NF-kB-dependent target gene expression.

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Objective. Aberrant splicing is one of the most significant components generating functional diversity in many
pathological conditions. The objective of this study was to analyse the mutations or aberrant splicing of A20
transcript, the region encompassing the ovarian tumour (OTU) domain [which is functionally important as an
inhibitor of nuclear factor (NF)-kB activation] in fibroblast-like synoviocytes (FLSs) from RA patients.

Hyun Kyung Yoon et al.

Materials and methods

Isolation of primary human RA FLSs and cell culture
RA FLSs were isolated by the enzymatic digestion of synovial tissues obtained from 10 RA patients undergoing total
joint replacement surgery or knee synovectomy at
Chungnam National University Hospital, Daejeon, Korea.
This study was approved by the Chungnam National
University Hospital institutional review board (approval

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number 1012-164) according to the Declaration of Helsinki

and written informed consent was obtained from each patient prior to surgery. After discarding fat and fibrous tissue,
the synovium was minced into small pieces and treated for
2 h with 2 mg/ml of type II collagenase in Hanks balanced
salts solution (HBSS) at 37 C in 5% CO2. The tissue was
then filtered using fine sterile gauze, washed and resuspended in DMEM supplemented with 10% FBS, 100 U/ml
penicillin and 100 mg/ml streptomycin. Dissociated cells
were then centrifuged at 800 g, then at 1000 g, and then
plated in 10-cm dishes. After overnight culture, the non-adherent cells were removed and the adherent cells were cultivated in DMEM supplemented with 10% FBS, 100 U/ml
penicillin and 100 mg/ml streptomycin. The cultures were
kept at 37 C in 5% CO2 and the medium was replaced
every day. When the cells approached confluence, they
were passaged into fresh culture dishes after trypsin/
EDTA treatment. RA FLSs from passages four to nine
were used in each experiment. The cells were morphologically homogeneous and exhibited the appearance of FLSs,
with typical bipolar configuration under inverse microscopy.
HEK293 cells were cultured in DMEM supplemented with
10% (v/v) fetal bovine serum, 2 mM glutamine, 100 U/ml
penicillin and 100 mg/ml streptomycin at 37 C in a humidified
atmosphere of 95% air and 5% CO2.

RTPCR analysis
Expression of the OTU domain of A20 in FLSs from 10 RA
patients was analysed by semi-quantitative RTPCR analysis. After the total RNA was prepared using a TRIZOL
reagent (Life Technologies, Inc.), oligo(dT)-primed cDNA
was synthesized using an RTPCR kit (Stratagene, La
Jolla, CA, USA). PCR amplification was carried out using
the primer pair specific to human A20 (50 -TGGCTGAACAAG
TCCTTCCT-30 and 50 -CTTCAGGGTCACCAAGGGTA-30 )
and human glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) (50 -GACCCCTTCATTGACCTC-30 and 50 -GCCAT
CCACAGTCTTCTG-30 ). After PCR amplification, the products were separated by agarose gel electrophoresis and
visualized using ethidium bromide staining.

A20 transcript sequencing

To sequence A20 transcripts in five representative FLSs, 5 mg
of RNA were reverse transcribed into the N-terminal region
encompassing the entire OTU domain of cDNA and cloned
using a T-Blunt PCR cloning kit (Solgent, Daejeon, Korea),
according to the manufacturers instructions. To sequence
the amplified cDNA bidirectionally, we used universal M13
forward and reverse primers (50 -GTAAAACGACGGCCAG
-30 ; 50 -CAGGAAACAGCTATGAC-30 ). At least 10 clones
were randomly selected, and the purified cDNAs were
sequenced using a BigDye Terminator v1.1 Cycle
Sequencing kit (Applied Biosystems, Foster City, CA, USA).
All mutations were confirmed on independent PCR products and analysed by alignment of each cDNA sequence
to its respective genomic sequence of A20 (GenBank accession no. NT025741).

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two specific serine residues, which is followed by its ubiquitination and proteasomal degradation, thereby releases NF-kB proteins and allows for their nuclear
translocation [5]. Constitutive activation of NF-kB regulates expression of multiple pro-inflammatory cytokines
that play an essential role in the pathogenesis of RA
[912].
As one of the NF-kB target genes, A20 [also known as
TNFAIP3 (TNF-induced protein 3)] has been well established for its negative feedback mechanism to block
NF-kB activation through its ubiquitin-editing function in
response to various inflammatory signalling, including
TNF, IL-1b and lipopolysaccharides [1316]. In the
TNF-mediated NF-kB signalling pathway, A20 eliminates
Lys63-linked ubiquitin chains from RIP1 with its
N-terminal ovarian tumour (OTU) domain and attaches
Lys48-linked polyubiquitins to RIP1 with the C-terminal
zinc finger (ZnF)-containing domain, thereby targeting
this factor for proteasomal degradation [17]. Mice null
for A20 develop severe multiorgan inflammation, including
inflammation of synovial joints, due to prolonged NF-kB
activation [18]. Multiple variants within chromosome 6q23,
which encodes for A20, has been associated with the
pathogenesis of a number of diseases, particularly autoimmune diseases and certain types of cancer. Inactivation
of A20 by the typical two mechanisms, deletions and inactivation mutations, has been reported in marginal zone
lymphomas, Hodgkins lymphoma, primary mediastinal
B cell lymphoma and activated B cell-like diffuse large
B cell lymphoma, which may contribute to lymphomagenesis [19, 20]. Also, multiple polymorphisms in the A20 are
closely associated with many pathological conditions,
including SLE, coronary artery disease in type 2 diabetes,
psoriasis and RA [2124]. Moreover, in our previous
study, in vivo experiments in CIA using A20 to block
the NF-kB pathway efficiently inhibited inflammatory
response and bone destruction [25], indicating the critical
role of A20 in RA pathophysiology. However, aberrantly
spliced variant forms of A20 have not yet been discovered
in RA.
Since the N-terminal OTU domain of A20 plays an essential role as a negative feedback inhibitor of NF-kB activity,
we analysed the mutations or aberrant splicing of the A20
transcript region encompassing the OTU domain in FLSs
from RA patients. In the present study, we discovered the
existence of four novel spliced forms, which were aberrantly encoded into the truncated OTU domain. These aberrantly spliced variant forms of A20 may function to
dysregulate NF-kB, leading to the pathogenesis of RA.

Intron-derived aberrant splicing of A20 gene in RA

Plasmid construction of variant forms of cDNA and

transfection
Wild-type and splice-variant forms of A20 cDNA were
subcloned into pCMV-Tag2b (Stratagene), generating
flag-A20/WT, flag-A20/INS-pI2, flag-A20/INS-I4, flagA20/Del-E4/INS-I4 and flag-A20/INS-pI2/Del-E4/INS-I4
with an N-terminal flag tag. All constructs were verified
to confirm the integrity of the inserts by DNA sequencing.
To detect the protein expression, HEK293 cells were transiently transfected either with the wild type or with the
variant forms of A20 using Lipofectamine PLUS reagent
by following instructions provided by the manufacturer
(Invitrogen, Carlsbad, CA, USA), and immunoblotted with
and anti-flag antibody.

Immunoblot analysis

Quantitative real-time PCR

After FLSs were treated with TNF (15 ng/ml) at different
time points, cells were collected and RNA was extracted
with TRIZOL reagent (Life Technologies, Inc., Carlsbad,
CA, USA). Diluted RNA was analysed as described [26],
and primers were obtained using the Qiagen QuantiTect
Primer Assay kit: TNF (Hs_TNF_3_SG, catalog no.
QT01079561),
IL-6
(Hs_IL6_1_SG,
catalog
no.
QT00083720) and GAPDH (Hs_Gapd_2_SG, catalog no.
QT01192646). Real-time quantitative PCR was carried
out using an SYBR Green detection system (Applied
Biosystems). The PCR conditions were optimized as follows: 95 C for 10 min, and 40 cycles each of 95 C for 15 s,
60 C for 10 s, and a dissociation stage at the end of the
run from 60 C to 95 C. In order to detect aberrantly
spliced variants, primers for intron 2 and intron 4 were
designed: primer for intron 2, 50 -TTGCCAGTTTTGTCCT
CAGTTTC-30 and 50 -GCCTCAATGTGCTCGTTCGT-30 ;
primer for intron 4, 50 -GGAAACAGACACACGCAACTTTA
A-30 and 50 -CAAGGCATAAGGCTGAAAGCA-30 . All reactions were independently repeated at least three times
to ensure reproducibility of the results.

Luciferase reporter assay

HEK293 cells were co-transfected with p2xNF-kB-Luc,
pRSV-b-galactosidase and flag-tagged wild-type and splice
variant forms of A20 constructs as indicated in the figure legends. Twenty-four hours after transfection, cells were

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Results
Expression analysis of A20 mRNAs from FLSs of RA
Since A20 plays an essential role as a negative regulator
of NF-kB in the condition of inflammatory disease, the
level of A20 expression or the presence of functionally
inactive mutant of the A20 gene may have a close relationship with the progression of RA (for instance, hyperplastic growth of FLSs). Therefore the expression profiles
of A20 mRNA from 10 RA patients were analysed by
RTPCR using a pair of primers encompassing the sequence of the OTU domain, a functionally important inhibitor of NF-kB activation. As expected, we detected a
major RTPCR product that is predicted to be 791 bp from
the published A20 cDNA sequence (GenBank accession
no. NM006290). Interestingly, additional larger fragments
were observed in 7 of the 10 FLSs compared with that of
HEK293 cells, which harbour ectopically expressed
wild-type A20 cDNA (Fig. 1). These results suggest the
possibility that aberrant transcript of A20 might be
widely expressed FLSs of RA.

Identification of four novel aberrant splice variants

in the OTU domain of A20 from RA FLSs
To identify any novel splicing in A20 mRNA, N-terminal
regions encompassing the entire OTU domain of A20
cDNA in five representative FLSs were individually
cloned and at least 10 clones of each cDNA sample
were examined for potential alterations at the transcriptional level by clone sequencing. Analysis of A20 cDNA
sequence revealed that the expression of A20 mRNA was
present in all five RA patients examined and their sequence integrity was well maintained (data not shown).
However, a total of 23 clones of A20 transcript variants
in addition to wild-type A20 transcripts were detected
and categorized into four aberrantly spliced variant
forms (Table 1). These aberrant transcripts included insertion and/or deletion of one or more region of the A20 gene.
A summary of these sequence alterations and diagrams
of the aberrant splicing are presented in Fig. 2.
Specifically, we found a partial insertional mutation
(50 bp) of intron 2 at the junction between exon 2 and
exon 3 (A20/INS-pI2; Fig. 2A, S1A), and another insertion
of intron 4, reserving the whole sequence of intron 4 (160
bp) at the junction between exon 4 and exon 5 (A20/
INS-I4; Fig. 2B, supplementary Fig. S1B, available as supplementary data at Rheumatology Online). We also found
a mutation at position 553700, deleting the whole of exon
4 and inserting the whole intron 4 instead (A20/Del-E4/
INS-I4; Fig. 2C, supplementary Fig. S1C, available as supplementary data at Rheumatology Online) as well as additional partial insertion of intron 2 with A20/Del-E4/INS-I4
(A20/INS-pI2/Del-E4/INS-I4; Fig. 2D, supplementary Fig.
S1D, available as supplementary data at Rheumatology

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After treatment with TNF (15 ng/ml) as described in the

figure legends, FLSs were collected and lysed in M2
buffer (20 mM Tris at pH 7.6, 0.5% NP-40, 250 mM NaCl,
3 mM ethylenediaminetetraacetic acid (EDTA), 3 mM ethylene glycol tetraacetic acid, 2 mM dithiothreitol 0.5 mM
phenylmethanesulphonylfluoride, 20 mM b-glycerol phosphate, 1 mM sodium vanadate, 1 mg/ml leupeptin). Fifty
micrograms of the cell lysates were subjected to SDS
polyacrylamide gel and blotted onto a polyvinyl difluoride
membrane. After blocking with 5% skim milk in phophatebuffered salineTween 20, the membrane was probed with
the relevant antibody and visualized by enhanced chemiluminescence (ECL), according to the manufacturers
instructions (Amersham, Piscataway, NJ, USA).

treated with TNF (15 ng/ml) for an additional 10 h and luciferase activities were measured using a luciferase assay kit
(Promega, Madison, WI, USA). Luciferase acivity was normalized relative to the b-galactosidase activity of each sample.

Hyun Kyung Yoon et al.

FIG. 1 mRNA expression analysis of the N-terminal region encompassing the OTU domain of A20 in RA FLSs.

RNA was isolated from 10 RA FLSs and HEK293 cell transfectant control of the human A20 gene. RTPCR was
performed with primers specific to human A20 and GAPDH. After the PCR amplication, the products were separated by
agarose gel electrophoresis and visualized using ethidium bromide staining. Normal-sized and larger transcripts were
present in RA FLSs, whereas only normal-sized transcript could be detected in the HEK293 cell-transfected A20 gene.

Splice event
A20/INS-pI2
A20/INS-I4
A20/Del-E4/INS-I4
A20/INS-pI2/Del-E4/INS-I4

Nucleotide change
Insertion of 50 bp (partial intron 2) between exon 2 and exon 3
Insertion of 160 bp (entire intron 4) between exon 4 and exon 5
Deletion of exon 4 (553700)
Insertion of 160 bp (entire intron 4) between exon 3 and exon 5
Insertion of 50 bp (partial intron 2) between exon 2 and exon 3
Deletion of exon 4 (553700)
Insertion of 160 bp (entire intron 4) between exon 3 and exon 5

Total

Online). The result of deduced amino acid sequence alignment was thought to produce various abnormal A20 proteins by frame shift translation (Fig. 2E and F). These
aberrant variants generated the premature termination
codon at the intron-derived sequence and might produce
truncated forms of A20, which disrupts the structure of the
N-terminal region of A20, including the OTU domain. A
BLAST (National Center for Biotechology Information)
search of the inserted nucleotides revealed 100% identity
with an A20 genomic DNA sequence in intron 2 and intron
4 (GenBank accession no. NT025741, nt 42362336
42362385 and nt 4236643042366589). These four types
of alteration were found singly or in combination (i.e. deletion of exon 4 and/or insertion of partial intron 2 and
whole intron 4) in five FLSs from RA patients examined.
Based on the sequence alignment of the variant A20
transcript, the mutational mechanism is probably an aberrant RNA splicing event. In particular, the TTGCAG sequence immediately upstream of the inserted intron 2 is a
perfect match to the YYNCAG consensus splice acceptor
sequence, while the GTGATT sequence immediately
downstream of the insert has only a single nucleotide mismatch compared with the canonical GTRAGT consensus
splice donor sequence (Fig. 3A). In addition, deletion of
exon 4 and insertion of intron 4 is also homologous to
consensus splice acceptor and donor sequence, since

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No. of events (%)

3 (13.0)
2 (8.7)
14 (60.9)
4 (17.4)

23 (100)

the TTTCAG sequences immediately at the end of exon

4 are well matched to consensus splice acceptor sequence (YYNCAG) (Fig. 3B). Collectively these data suggested that the efficiency of mRNA splicing might be
affected without nucleotide substitutions, leading to the
formation of aberrant A20 transcript in FLSs from RA
patients.

Loss of NF-kB inhibitory function by aberrantly

spliced variants of A20
As A20 is a key regulator of NF-kB signalling that negatively
modulates NF-kB activation through a variety of receptors
including TNF receptor [17], we investigated the function of
these aberrantly truncated forms of A20 proteins. To confirm the protein expression of aberrant variants of A20 transcripts, HEK293 cells were transiently transfected with
flag-tagged wild-type and four novel variants (A20/
INS-pI2, A20/INS-I4, A20/Del-E4/INS-I4, A20/INS-pI2/
Del-E4/INS-I4) of A20. As shown in Fig. 4A, we detected
the expression of various truncated sizes of A20 proteins
predicted to be 11.9, 27.5, 21.3 and 11.9 kDa, which correlates well with their corresponding mRNAs. We then further examined whether these truncated forms of A20 affect
TNF-induced NF-kB activation using NF-kB luciferase reporter plasmid bearing the NF-kB-binding DNA sequence.
As consistent with previous reports [17], ectopic

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TABLE 1 Aberrant spliced variant forms of A20 in FLSs from RA patients

Intron-derived aberrant splicing of A20 gene in RA

FIG. 2 Aberrant splice variants of A20 transcripts expressed in RA FLSs.

over-expression of wild-type A20 almost completely

suppressed TNF-induced NF-kB activation. In contrast,
over-expression of four RA FLS-derived A20 mutants
(A20/INS-pI2, A20/INS-I4, A20/Del-E4/INS-I4, A20/INSpI2/Del-E4/INS-I4) failed to show down-regulation of
TNF-induced NF-kB activity (Fig. 4B), indicating that
these were actually loss-of-function mutations.

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Aberrant A20 transcript enhanced NF-kB signalling in

FLSs from RA patients
To detect the variant-specific mRNA transcript in the
conditions of FLSs from RA patients, real-time RTPCR
was performed with one primer selected within inserted
intron 2 or intron 4 sequence and the other from a

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(A) A20/INS-pI2 exhibits the partial insertional mutation of intron 2 (50 bp) at the junction between exon 2 and exon 3.
(B) A20/INS-I4 exhibits the insertional mutation of intron 4 (160 bp) at the junction between exon 4 and exon 5. (C) A20/
Del-E4INS-I4 skipped exon 4 and contained an insertional mutation of the entire intron 4 (160 bp) at the junction between
exon 3 and exon 5. (D) A20/INS-pI2/Del-E4/INS-I4 contained an additional partial insertional mutation of intron 2 (50 bp) with
A20/Del-E4INS-I4. (E) Diagrammatic representation of the A20 domain and amino acid number in GenBank cDNA clone
NM006290. Two conserved domains are indicated by grey-coloured boxes, including an OTU domain; and the seven zinc
finger (ZF) domains. (F) Amino acid sequence alignments of aberrantly spliced variant forms of A20 were found in RA FLSs.
Uppercase letters represent a reference sequence of wild-type A20, while underlined lowercase letters indicate sequence
variants due to a frame shift in translation (WT: A20/wild-type; MT-1: A20/INS-pI2; MT-2: A20/INS-I4; MT-3: A20/Del-E4/
INS-I4 and MT-4: A20/INS-pI2/Del-E4/INS-I4). The asterisk indicates a stop codon.

Hyun Kyung Yoon et al.

FIG. 2 Continued.

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Discussion
Eukaryotic genes characteristically produce large amounts
of pre-mRNA-containing introns, which are removed by a
highly accurate cleavage/ligation reaction known as splicing. Nonetheless, aberrant splicing is one of the most significant components generating functional diversity in many
pathological conditions, such as tumorigenesis and autoimmune disease [27, 28]. In this study we provided clear
evidence that four types of novel aberrant splicing transcript of A20 occur in FLSs from RA patients. The fact
that these aberrant splicing variants result in frame shift,
stop translation and eventually OTU domain (which targets
RIP1 or TRAF6 de-ubiquitination) disruption of A20 indicates that these novel forms of spliced variants could contribute to the dysregulation of NF-kB activation status in RA
FLSs. These data suggest the important role of aberrant
splicing of A20 in the aetiology and/or progression of RA.
Although the aetiology of RA is still not understood,
accumulating evidence largely indicates that transactivation of NF-kB-dependent gene expression plays a key role
in the development of RA and many other autoimmune
diseases. Indeed, aberrant dysregulation of NF-kB activity
or an increased nuclear level of the NF-kB subunit has
been commonly observed in cultured FLSs, human arthritic joints and joints of animals with experimentally
induced RA [912]. Consequently, selective targeting of
the products of NF-kB-driven genes, such as TNF, IL-6
and IL-1, has been emerging as a valuable breakthrough
in the treatment of RA patients who do not respond to
standard treatment [29]. To achieve tight regulation of

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remote exon encompassing the corresponding intron.

This approach revealed that the levels of expression
of variant forms of A20 varied among 10 distinct FLSs
(Fig. 5A). We then proceeded to examine whether the
splice variant forms of A20 indeed affect the status of
NF-kB activity. To address this issue, we compared the
extent of TNF-induced phosphorylation of IkB-a in FLSs
that harbour different levels of aberrant A20 transcript
(sample FLS #1 vs sample FLS #4). As expected, TNF
treatment resulted in rapid and progressive induction of
A20 by a monoclonal antibody, which only recognizes
the full length of wild-type A20 protein in both FLS #1
and FLS #4 (Fig. 5B, top panel). Notably, the basal and
TNF-induced expression level of A20 in FLS #4 carrying
the highest variant forms of A20 was much lower than
that of FLS #1. Furthermore, when FLS #1 was treated
with TNF, the phosphorylation of IkB-a was transiently
enhanced by 1 h, after which the level continued to decrease gradually. In FLS #4, however, the TNF-induced
phosphorylation of IkB-a was not only greatly enhanced
but also persistent (Fig 5B, middle panel). These results
suggest that the level of aberrant A20 transcript affects
the NF-kB signalling pathway in response to TNF.
To gain further insight into the consequence of NF-kB
signalling in the same conditions, we assessed
NF-kB-dependent target gene expression by quantitative
real-time PCR. Consistently the transcription of TNF and
IL-6 were more drastically up-regulated after TNF treatment in FLS #4 (Fig 5C), which correlated with the
enhanced phosphorylation status of IkB-a (Fig. 5B,
middle panel).

Intron-derived aberrant splicing of A20 gene in RA

FIG. 3 A schematic representation of aberrant splicing forms of the A20 gene.

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(A) The 50 bp of intron 2 sequence that is inserted between exon 2 and exon 3, is shown in a shaded box. Consensus
splice donor (ttgcag) and acceptor (gtgatt) sequences flanking this 50-bp insert are depicted as bold. The aberrant
cleavage sites within intron 2 are indicated by the arrow. (B) A20 genomic sequence showing the deletion of exon 4 and
insertion of entire intron 4. The universally conserved motifs near the nucleotide sequences surrounding the exonintron
borders that act as essential splicing signals are depicted as bold. Arrows indicate aberrant cleavage sites for exon 4
deletion and the nucleotide sequnces of the entire intron 4 that are inserted into the mutant A20 are shown in the
shaded box.

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Hyun Kyung Yoon et al.

FIG. 4 Effects of ectopic expression of wild-type and

aberrantly spliced variants on TNF-induced NF-kB
activation.

NF-kB signalling, a number of cells employ different control mechanisms to keep NF-kB signalling in check [15].
Functioning as a gatekeeper, A20 has a pivotal role in the
restriction of the duration of both TNF and Toll-like receptor-mediated NF-kB signals, and thus has been described

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(A) Wild-type and various truncated forms of A20 were

transfected into HEK293 cells, and the expression was
determined by immunoblotting with anti-flag antibody
(top panel). The blot was verified using an antibody
against b-actin to confirm equal amounts of proteins were
loaded (bottom panel). (B) HEK293 cells were transiently
transfected with expression vectors of wild-type and
various truncated forms of A20 constructs along with
p2xNF-kB-Luc and pRSV-b-gal. After 24 h of transfection,
cells were treated for 10 h with 15 ng/ml of TNF. Cells were
then lysed and the luciferase activities were measured
as described in the Materials and methods section.
The luciferase activity of each sample was normalized
according to b-galactosidase activity. Results show the
mean (S.E.) of at least three independent experiments.

as a potent anti-inflammatory molecule [1316]. Based on

the recent data using genome-wide association studies
(GWASs), variants in the A20 gene have been shown to
be associated with multiple inflammatory and autoimmune diseases, including RA, SLE, Crohns disease and
psoriasis [2224, 30], which suggests that a defect in A20
expression or activity may be involved in the pathogenesis
of these diseases. Moreover, A20 was frequently inactivated by somatic mutation or deletion in several subsets
of B-cell lymphomas [19, 20], suggesting that the genetic
variants of A20 also function as a tumour promoter in certain types of tumour.
In addition to the mutations and chromosomal aberrations of A20 described previously, we identified here the
four types of aberrant splicing transcript of A20 in RA
FLSs: (i) 50 nt in the middle of intron 2 inserted out-frame
between exon 2 and exon 3 (A20/INS-pI2); (ii) 160 nt of the
entire intron 4 inserted between exon 4 and exon 5 (A20/
INS-I4); (iii) exon 4 deleted in A20/INS-I4 (A20/
Del-E4INS-I4); (iv) additional partial intron 2 (50 nt) inserted
in A20/Del-E4INS-I4 (A20/INS-pI2/Del-E4/INS-I4). In our
study, in the most abundant aberrantly spliced form
(A20/Del-E4/INS-I4) exon 4 was skipped and intron 4
inserted, which introduced premature translation
termination codon, resulting in 192 amino acids of protein
(Table 1, Fig. 2). Three other minor A20 variantsA20/
INS-pI2, A20/INS-I4 and A20/INS-pI2/Del-E4/INS-I4
also produced truncated proteins 108, 248 and 108 in
amino acid size, in contrast to the 790 amino acids of
full-length A20 protein (Fig. 2). Although the precise mechanism of aberrant splicing in pathological conditions such
as autoimmune disease and cancer is still unknown, the
common reason for splicing defects is a point mutation in
the genomic splice site [31, 32]. To access whether the
aberrant transcripts of A20 were caused by genomic
sequnce mutation, PCR was used to amplify genomic
DNA across the regions where the aberrant splicings
occurred. In FLSs containing the aberrant A20 transcripts,
we did not detect any mutation or deletion of the A20 gene
at the genomic level, especially within the cis-acting consensus sequence of intron/exon boundaries (data not
shown). In contrast, 50 and 30 splice sites in A20 aberrant
transcripts obeyed the GT/AG rule, and the branch sites
matched well with consensus sequence (Fig. 3), indicating
that the aberrant splicing of A20 in RA FLSs was not a
result of mutations in cis-acting consensus sequence at
the potential cryptic donor and acceptor sites. In a recent
GWAS, it was reported that several SNPs at 6q23 were
associated with RA susceptibility [33, 34]. Given that
among these RA-associated SNPs, rs5029937 in particular is located in the intron 2 of A20, the generation of such
novel aberrant transcripts may be affected, especially
the insertion of partial intron 2 (A20/INS-pI2 and/or
A20/INS-pI2/Del-E4/INS-I4) in our study. However, the
rs5029937 risk allele is not only located >2.2 kb from the
aberrant splicing site within intron 2 but more importantly,
it does not contribute to the generation of consensus
splice donor/acceptor sequence (GT/AG). Thus it seems
that the presence of an allele of rs5029937 and aberrant

Intron-derived aberrant splicing of A20 gene in RA

FIG. 5 The level of aberrant transcripts of A20 correlates with persistent activation of NF-kB signalling in FLSs from RA.

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(A) After total RNA was isolated in FLSs from 10 RA patients, the relative expression levels of the aberrant transcripts of
A20 were analysed using the primer pair specific to intron 2 or 4 by real-time RTPCR and normalized according to
b-actin mRNA levels. Results show the mean (S.E.) of at least three independent experiments. (B, C) FLSs from two RA
patients (RA FLS #1 vs RA FLS #4) were treated with TNF (15 ng/ml) for various times as indicated. Whole-cell extracts
were subjected to immunoblotting with anti-A20, anti-pIkB-a and anti-actin antibodies (C). The relative transcription
levels of NF-kB target genes, including TNF-a and IL-6, were measured by real-time RTPCR as described in the
Materials and methods section. Results show the mean (S.E.) of at least three independent experiments.

splicing transcripts of A20 are independently associated

with RA susceptibility. Nonetheless, since it has been proposed that the combination of independent genetic variants at the intergenic region encompassing the
RA-associated SNPs at 6q23 can increase the risk estimate for RA [35], future large-scale genetic studies are

www.rheumatology.oxfordjournals.org

required to ascertain the association of the identified

SNPs and aberrant splicing variants of A20 in the progression of RA.
Although the reason why A20 transcripts are defective
in FLSs from RA patients remains unclear, accumulating
evidence suggests that certain lineages of cells such as

435

Hyun Kyung Yoon et al.

Rheumatology key messages

Intron-derived aberrant splicing variants of A20
occur exclusively in FLSs from RA patients.
. A20 spliced variants contribute to dysregulation of
NF-kB activation status in RA FLSs.
. Aberrant splicing of A20 might be associated with
the aetiology and/or progression of RA.
.

Acknowledgements
We thank Ji Hoon Lee (Amherst College, Amherst, MA,
USA) for assistance in the preparation of this manuscript.

436

Funding: This work was supported by the National

Research Foundation of Korea (NRF) through a grant
funded by the Korean government (MEST) (no.
2012-0005764), (KRF-2009-352-E00009), Chungnam
National University Hospital Research Fund 2010 and a
grant from the National R&D Program for Cancer Control
Ministry of Health & Welfare, Repubic of Korea (no.
0720560). This study was also supported by a grant
(A070001) from the Korean Health Technology R&D
Project, Ministry of Health & Welfare, Republic of Korea.
Disclosure statement: The authors have declared no conflicts of interest.

Supplementary data
Supplementary data are available at Rheumatology
Online.

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