Archives of Biochemistry and Biophysics 523 (2012) 151–160

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Expression profile and interactions of hnRNP A3 within hnRNP/mRNP complexes

in mammals
Christina Papadopoulou, Georgios Boukakis, Vassiliki Ganou, Meropi Patrinou-Georgoula,
Apostolia Guialis ⇑
RNA Processing Program, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Avenue, 11635 Athens, Greece

a r t i c l e i n f o a b s t r a c t

Article history: The hnRNP A/B family contains abundant nuclear proteins with major roles in alternative splicing and the
Received 28 November 2011 ability for nucleo-cytoplasmic shuttling. Compared to the best known members of this family (hnRNP A1,
and in revised form 27 March 2012 A2/B1), hnRNP A3 is a relatively less known protein. We report herein immunochemical studies with the
Available online 22 April 2012
hnRNP A3 isoforms (A3a and A3b) that provided evidence for species-specific expression. The unspliced
A3a was found in human and murine cells, while the spliced A3b was a unique and abundant isoform in
Keywords: mouse/rat. In addition, a tissue-specific variation was observed in mice, as the brain was the only tissue
hnRNP A3
found to overexpress hnRNP A3a. Both hnRNP A3a and A3b were able to stably associate with immunos-
hnRNP A/B proteins
hnRNP/mRNP complexes
elected hnRNP and mRNP complexes. Use of the auxiliary domain of hnRNP A3 in pull-down assays on
Protein–protein interactions human cell extracts revealed its unique ability to form a network of interactions not only with other
Post-transcriptional control A/B proteins but also with additional hnRNPs. All interactions, except those of hnRNP A1, were highly
enhanced by previous RNase A digestion of the extracts. Our findings revealed novel characteristics of
hnRNP A3 and supported its extensive involvement in the many aspects of mRNA maturation processes
along with the other hnRNP A/B proteins.
Ó 2012 Elsevier Inc. All rights reserved.

Introduction protein–protein interactions. These proteins all contain a combina-

Proteins with the ability to bind hnRNA1 (pre-mRNA) and mRNA
constitute a large group of RNA-binding proteins (RBPs) with a major
role in the post-transcriptional regulation of RNA pol II genes. In par-
ticular, hnRNP proteins represent a special group of RBPs that are
stable components of hnRNP complexes, the sites of mRNA matura-
tion. More than 20 distinct hnRNP proteins have been identified,
ranging from 32 to 110 kDa and designated (in order of increasing
molecular weight) as hnRNP A–U [1]. The majority of hnRNPs con-
tain alternatively spliced variant species as well as a number of
post-translational modifications (mainly phosphorylation and argi-
nine methylation). In addition to their generalized role as RNA pack-
aging proteins, hnRNPs have specialized functions that are
dependent on their ability to form specific RNA-protein and
⇑ Corresponding author. Fax: +30 210 7273755.
E-mail address: aguial@eie.gr (A. Guialis).
1
Abbreviations used: hnRNP, heterogeneous nuclear ribonucleoprotein; RBP, RNA-
binding protein; RRM, RNA recognition motif; RBD, RNA binding domain; KH, K
homology; RGG, Arg-Gly-Gly box; CBF-A, CArG box-binding factor-A; A2RE, A2
response element; 30 UTR, 30 untranslated region; 2-D, two-dimensional; NEPHGE,
non-equilibrium pH gradient electrophoresis; SDS–PAGE, SDS–polyacrylamide gel
electrophoresis; GST, glutathione S-transferase; IPTG, isopropyl b-D-1-thiogalactopy-
ranoside; ECL, enhanced chemiluminescence; PAS, protein A-sepharose; ssDNA,
single-stranded DNA; IP, immunoprecipitation.
tion of known RNA-binding domains referred to as the RRM/RBD
(RNA recognition motif or RNA-binding domain), the KH (K homol-
ogy) motif and the RGG (Arg-Gly-Gly) box, with the latter also func-
tioning in protein–protein interactions [2]. The RRM/RBD is the most
common and best analyzed motif both structurally and functionally
[3]. The hnRNP proteins have a major nucleoplasmic localization
with several of them (A, D, E, I, K, L) capable of nucleo-cytoplasmic
shuttling, while others (C and U) do not exit the nucleus except un-
der certain cellular conditions [4,5]. In addition to the major nuclear
role of hnRNPs in mRNA processing (splicing, polyadenylation) and
transport, they are known to participate in several other events,
including transcription, DNA repair and telomere DNA formation.
The limited cytoplasmic localization of some hnRNPs is also of func-
tional importance and affects the stability, translation and localiza-
tion of their associated mRNAs. Thus, hnRNPs are considered
important gene regulators, and any alteration in their expression
pattern is expected to affect many cellular functions (for recent re-
views, see [6,7]).
In particular, the members of the hnRNP A/B-type family (A1,
A2/B1, A3, and A0) constitute a special family of proteins that share
sequence and structural similarity. These proteins have two N-ter-
minal RRM/RBD domains in tandem followed by a C-terminal aux-
iliary domain that is rich in glycine (2xRBD-Gly). Their high
sequence homology is mainly due to their shared N-terminal

0003-9861/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.abb.2012.04.012
152 C. Papadopoulou et al. / Archives of Biochemistry and Biophysics 523 (2012) 151–160

RNA-binding domain, while their functional diversity originates
from the less conserved auxiliary domain that is mainly responsi-
ble for protein–protein interactions [8,9]. Additional members of
the hnRNP A/B family of murine and human origin, referred to as
the D-subgroup (hnRNP AB, D, DL), are also known [10]. A tran-
scriptional regulator belonging to this subgroup, named CBF-A
(CArG box-binding factor-A), has been identified as the mouse
orthologue of the human hnRNP AB [11] and has since attracted
a great deal of interest [12].
The hnRNPs A/B and C1/C2 are highly expressed hnRNPs that,
together with their associated RNAs, compose stable components
of the 40S core particles as isolated from partially degraded nuclear
extracts [13]. The most related A/B proteins are hnRNP A1, A2/B1
and A3, the latter being a relatively new and less studied member.
The participation of hnRNP A1 (the prototype A/B-type protein)
and A2/B1 in many cellular functions has been documented by a
series of studies. These proteins primarily act as splicing regulators
(usually splicing repressors), thus affecting the expression of their
associated mRNA targets [9,14 for Refs. therein]. Downstream
mRNA targets involved in a variety of cellular functions have been
determined for hnRNP A1, A2 and A3 in human cells, and a signif-
icant number of which are shared among these hnRNPs [9]. All
three proteins are, together with additional hnRNPs and other
RBPs, members of a group of proteins able to associate with the
AU-rich cis-elements in the 30 UTR regions of many mRNAs and
contribute to their stability and translation [15,16]. Other func-
tions shared by hnRNP A2/B1 (but not A1) [17], A3 [18] and CBF-
A [19] include their ability to act as mRNA trafficking trans-factors
and to directly bind a 21-nucleotide-long element, called the
A2-response element (A2RE), that is present in several mRNAs of
neural cells. Moreover, hnRNP A1, A2/B1 and A3 bind to both sin-
gle-stranded telomere DNA and telomerase RNA, regulating their
interactions [20–22].
Our interest in hnRNP A3 arose from the detection of autoanti-
bodies that react against discrete hnRNP A/B-type proteins in the
sera of patients with systemic autoimmune diseases. A specific
class of these autoantibodies targeted a major protein component
(named mBx) of the rat liver 40S hnRNP complexes, which was
nearly undetected in human (HeLa) cells [23]. The subsequent iso-
lation and characterization of a partial cDNA clone coding the en-
tire auxiliary domain of the rat mBx identified it for the first
time as the murine-specific hnRNP A3 protein [24]. We have since
referred to this hnRNP A/B protein that is abundant in murine cells
as hnRNP A3/mBx [25,26]. Thereafter, Ma et al. [18] reported the
isolation from rat and human brain of a full-length cDNA encoding
hnRNP A3 that, with the exception of one amino acid, had an iden-
tical sequence between human and rat species. This isolation led to
the characterization of the two main variants: a 41 kDa unspliced
form that incorporates the N-terminal exon consisting of 22 amino
acids (hnRNP A3a) and its 38 kDa spliced variant (hnRNP A3b)
[18,27]. An apparent functional redundancy between hnRNP A3
and A1 has been suggested based on their high sequence similarity
[18] as well as on RNAi suppression studies [28].
The present study focused on the immunochemical character-
ization of hnRNP A3 in mammals with the aim to describe novel
biochemical properties that might relate to its biological function.
Aided by the production of a homemade antibody able to react
with both the unspliced and spliced forms of hnRNP A3 (A3a and
A3b, respectively), we have verified their differential expression
patterns in human and murine cells. We also investigated the tis-
sue-specific expression of the hnRNP A3 isoforms in mice. The ma-
jor isoform in all examined mouse tissues was hnRNP A3b, with the
notable exception of the brain, in which the unspliced A3a isoform
was overexpressed. In addition, the combination of immunopre-
cipitation studies and pull-down assays using the GST-fused auxil-
iary domain of hnRNP A3 revealed novel characteristics of hnRNP
A3 that support its extensive participation in a network of interac-
tions for the assembly of hnRNP complexes.
Materials and methods
Cell culture and extract preparation
Two human cell lines, HeLa (cervix carcinoma) and A549 (lung
adenocarcinoma), were used interchangeably to prepare cell ex-
tracts. Monolayer cultures of exponentially growing cells were
maintained in D-MEM medium with 10% FCS. The rat Novikoff hep-
atoma cell line was similarly grown in D-MEM. Additionally, three
mouse cell lines (LA-4, CMT64/61 and PVDC57) were employed.
The lung cell lines LA-4 (adenoma) and CMT64/61 (carcinoma)
were purchased from the European collection of cell cultures
(ECACC). The LA-4 cells were grown in Ham’s F12 medium supple-
mented with 2 mM glutamine, 1% non-essential amino acids, 15%
FCS, and CMT64/61 in Waymouth’s MB 752/1 medium with
2 mM glutamine and 10% FCS. The third mouse cell line of skin ori-
gin, PDVC57 (squamous cell carcinoma), was derived as described
in [29] and provided by VK Zoumpourlis (IBRB-NHRF). These cells
were grown in D-MEM with 10% FCS. Penicillin and streptomycin
(1% v/v) were added to all cultures. All culture media and FCS were
obtained from Gibco Co.
The preparation of the nuclear and cytoplasmic extracts from
cultured cells was performed as outlined previously [30]. Affinity
chromatography on ssDNA-cellulose beads was employed to purify
hnRNP proteins from whole cell extracts that had been previously
treated with micrococcal nuclease to remove nucleic acids [31].
The proteins that eluted from the beads were resolved on the
two-dimensional (2-D) non-equilibrium pH-gradient electrophore-
sis (NEPHGE/SDS–PAGE) gel system as described previously [26].
The whole cell extracts were obtained by lysing a cell pellet
containing approximately 1–2  107 cells/ml in NET-2 buffer
(10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.05% Nonidet P-40), in
the presence of 2 lg/ml protease inhibitor mix (Sigma Co.) on
ice. Following sonication (6, 20 s) and centrifugation at
10,000 rpm for 10 min at 4 °C, the resulting supernatant was col-
lected and stored at 80 °C.
Antibodies
The monoclonal antibodies targeting hnRNP A1 (4B10), hnRNP
A2/B1 (DP3B3) hnRNP C1/C2 (4F4), hnRNP L (4D11), hnRNP M
(1D8) and a-tubulin (TU-02) were purchased from Santa Cruz Bio-
tech. The monoclonal antibody against b-actin (1501) was pur-
chased from Chemicon. The antibody against CBF-A, which was
raised in rabbits as described in [19], was the kind gift of P. Percip-
alle (Sweden). The rabbit polyclonal antibody against hnRNP A3
[anti-A3(N)] that was raised against the N-terminal exon unique
to the unspliced hnRNP A3a variant was obtained from Abcam Lim-
ited, UK (code No. 50949).
The rabbit anti-A3aux antibody was produced in our lab by using
the E. coli-expressed auxiliary domain of hnRNP A3 fused to GST as
an immunogen. The recombinant GST-A3aux protein was first re-
solved by SDS–PAGE and localized on the gel by Coomassie staining.
The corresponding gel area was excised, pulverized in liquid nitro-
gen and homogenized in phosphate-buffered saline (PBS). The
immunization protocol was performed as reported in [32]. The ob-
tained rabbit serum was clarified by centrifugation at 6500g for
10 min at 4 °C, mixed with an equal volume of PBS and brought to
a final concentration of 50% in (NH4)2SO4. After stirring on ice for
6 h, the solution was centrifuged as described above, and the col-
lected pellet was resuspended in PBS and dialyzed extensively
against the same buffer. The affinity purification of the antibodies
 C. Papadopoulou et al. / Archives of Biochemistry and Biophysics 523 (2012) 151–160
contained in the dialyzed material was performed against the re-
combinant GST-A3aux protein that was first resolved by SDS–PAGE
and then immobilized on a nitrocellulose membrane. The elution of
the membrane-bound antibodies was performed as in [26].
Protein resolution and western blot analysis
The protein samples were resolved by SDS–PAGE (10% acrylam-
ide gel) and then transferred to nitrocellulose membranes. Wes-
tern blotting with the application of the appropriate antibody
dilution and the visualization of the antigen–antibody reaction
with ECL was performed as in [30].
Mouse tissue lysate preparation
Animal tissues were obtained from a male young adult Balb/c
mouse. The dissected tissues were immediately snap-frozen in li-
quid nitrogen and kept frozen until use.
For a total tissue lysate, a small piece (in the range of 50–60 mg)
was cut from the frozen tissue and homogenized in 5 vol. of
homogenization buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl,
1 mM EDTA, 1% Nonidet P-40 and 2 lg/ml protease inhibitor
mix; Sigma Co.) as described in [33]. The NE-PER nuclear and
cytoplasmic extraction reagents kit (Pierce Biotechnology) was
employed with weighted portions of liver (Li) and brain (Br) tissue
(50 mg each), and the corresponding nuclear and cytoplasmic ex-
tracts were isolated following the exact instructions provided by
the manufacturer in the presence of the protease inhibitor mix.
The concentration of the soluble protein corresponding to the
whole tissue lysates as well as of the brain and liver nuclear and
cytoplasmic extracts was determined by the Bradford assay.
Immunoprecipitation and pull-down assays
The application of the immunoprecipitation (IP) reactions was
performed as described previously [30]. Protein A-Sepharose
(PAS) beads were incubated with the appropriate antibody, and
the resulting PAS-IgG fraction was mixed with the cellular extract.
As a negative control, IP reactions were performed either with
beads alone (mock IP) or with mouse IgG of the same isotype (in
equivalent amounts to the antibody applied). The bound proteins
were released from the beads directly in SDS-sample buffer and re-
solved by SDS–PAGE followed by western blotting with appropri-
ate antibodies.
In vitro pull-down assays were performed as in [30] on nuclear
and cytoplasmic extracts that had been pre-cleared by incubation
with glutathione beads. The subsequent incubation of the cellular
extracts was performed with either GST or GST-fused A3aux re-
combinant proteins bound to glutathione beads. The resolution
of all of the proteins bound to the beads was performed as for
the IP reactions.
Construction and expression of recombinant proteins
The isolation of the partial cDNA clone encoding the auxiliary
domain of the rat hnRNP A3/mBx was previously described by
our lab [24]. To generate constructs for the expression of the cod-
ing auxiliary domain of hnRNP A3 (A3aux) fused to glutathione
S-transferase (GST), the cDNA was excised from a pBluescript plas-
mid and re-inserted into the expression vector pGEX4T3 (Amer-
sham). The fused GST-A3aux protein and GST alone were used to
transform the bacterial strain BL21(DE3), and their expression
was induced with isopropyl b-D-1-thiogalactopyranoside (IPTG).
Glutathione–agarose beads (Sigma) were then used to purify the
recombinant proteins from the bacterial lysates as described in
[34].
153
The expression of the full-length hnRNP M as a GST-fused pro-
tein and the subsequent cleavage of the GST tag were performed as
described in [34].
Results
Differential expression of hnRNP A3 isoforms in cells of human and
murine origin
To establish the previously described presence of discrete
hnRNP A3 isoforms in human and rat/mouse cells [23], we per-
formed a direct comparison of all hnRNP A/B protein species from
a human (HeLa) and a rat (Novikoff) cell line. Whole cell extracts
were subjected to affinity chromatography on ssDNA-cellulose col-
umns to greatly enrich for the hnRNP proteins that were then re-
solved by 2-D (NEPHGE/SDS–PAGE) gel electrophoresis and
transferred to a nitrocellulose membrane. The Ponceau S staining
of the resolved protein species is presented in Fig. 1A (upper part),
and the position of hnRNP A/B is marked with a square box. The
immunochemical identification of these proteins was performed
after the western blotting with monoclonal antibodies against
hnRNP A2/B1 or A1 as well as with the commercial rabbit antibody
raised against the N-terminal exon of the unspliced hnRNP A3a iso-
form [anti-A3(N)] (Fig. 1A). As observed, similar protein spots for
hnRNP A1, A2/B1 and A3 were detected in both cell lines. In addi-
tion, the parallel application of one of our previously characterized
human autoimmune sera with a mixed specificity against hnRNP
A2/B1 and the murine-specific hnRNP A3/mBx (denoted a-B2/Bx,
a-A2) [26] verified the presence within the Novikoff hnRNP A/B
proteins of at least three major spots corresponding to hnRNP
A3/mBx that were undetected in HeLa cells, in clear opposition
to the detection of hnRNP A2 in both cell lines (Fig. 1A).
The immunochemical identification of the hnRNP A/B proteins
presented in Fig. 1A permitted the labeling of almost every spot
in the HeLa and Novikoff hnRNP A/B blots, as shown in Fig. 1B. This
assignment in the rat (Novikoff) cell line was in perfect agreement
with our previous report on the rat liver hnRNP A/B proteins [26].
The application of two discrete anti-hnRNP A3 antibody specifici-
ties (rabbit polyclonal and human autoantibodies) provided the
first evidence for the differential expression of two hnRNP A3 iso-
forms: the hnRNP A3 isoform, which is common to both human
and mouse, and hnRNP A3/mBx, which is the major isoform unique
to murine cells. To simplify the hnRNP A3 protein labeling and to
conform with the recently reported major hnRNP A3 isoforms of
41 and 38 kDa [18], we shall hereafter refer to the isoform common
to human and mouse as the unspliced hnRNP A3a and to the major
murine isoform, hnRNP A3/mBx, as the spliced hnRNP A3b isoform.
With the exception of the commercially available rabbit anti-
body against hnRNP A3a, we had no access to a comparable anti-
body for hnRNP A3b, which would be a desirable tool for further
studies. As the use of the autoantibodies in human sera that reacted
with hnRNP A3 had limited application due to their polyspecific
nature, we produced a new antibody. For this, we utilized our pre-
viously reported partial cDNA clone of rat origin that codes for the
entire C-terminal auxiliary domain of hnRNP A3 that is common to
both isoforms [24] and expressed it as a GST-fused product (GST-
A3aux). The specificity of this new, affinity-purified rabbit antibody
(anti-A3aux) and its direct comparison with the commercial anti-
A3(N) (applied above) is shown in Fig. 2A in parallel with the immu-
nodetection of hnRNP A1, which served as a reference hnRNP A/B
protein, and b-actin. This comparison involved the western blotting
of whole cell extracts that were prepared from two human (HeLa
and A549) and three mouse (CMT, LA-4 and PVDC) cell lines. As ex-
pected (see also Fig. 1A), the application of the anti-A3(N) antibody
led to reaction with a single protein species common to both human
and mouse cell lines, which corresponded to the hnRNP A3a
154 C. Papadopoulou et al. / Archives of Biochemistry and Biophysics 523 (2012) 151–160

A
HeLa Novikoff
NEPHGE
kDa H+ OH- H+ OH-
B
116
SDS-PAGE

97 HeLa
80
66 B2
A1B
55 A3 (A3a)
B1
45

A2
A1
30

hnRNP A/B
WB: Novikoff
hnRNP B1
α-A2/B1
hnRNP A2 B2
A3/mBx (A3b)
A1B
A3 (A3a)
B1

α-A1 hnRNP A1B A2

hnRNP A1 A1

α-A3(N) hnRNP A3

AutoAbs:
hnRNP A3/mBx
α-B2/Bx, α-A2 hnRNP A2

Fig. 1. Direct comparison of the hnRNP A/B proteins between human (HeLa) and murine (rat Novikoff) cells. The hnRNP proteins were isolated from whole cell extracts by
affinity chromatography on ssDNA-cellulose columns, and equal protein amounts (100 lg) were resolved on 2-D NEPHGE/SDS–PAGE gels. (A) Following transfer to
nitrocellulose membranes, the resolved proteins were visualized by reversible staining with Ponceau S. The position of hnRNP A/B is indicated by a box. The membranes were
then western blotted using monoclonal antibodies against hnRNP A2/B1 and A1, the a-A3(N) rabbit antibody targeting hnRNP A3 and a human autoimmune serum with
mixed specificity for hnRNP A2 and the A3/mBx variant form exclusive to Novikoff. (B) The assignment of the immunochemically identified hnRNP A/B protein species,
including the unspliced hnRNP A3(A3a) and the spliced A3/mBx(A3b) isoforms, is indicated in the enlarged picture of the proteins boxed in A.

isoform. This reaction was clearly distinct from the specificity of the
anti-A3aux antibody, which recognized the hnRNP A3b isoform as
the major protein species almost exclusive to the mouse cell lines
and also reacted with the hnRNP A3a isoform common to both hu-
man and mouse. An unexpected finding with the anti-A3aux anti-
body was the reaction with an additional protein of
approximately 75 kDa (and a probable degraded fraction of it) that
was unique to the mouse cell extracts (Fig. 2A). As this antibody
population was affinity purified against the same recombinant pro-
tein used as an immunogen, this presently unidentified cross-reac-
tive murine-specific protein is likely to share some degree of
homology with the auxiliary domain of hnRNP A3.
Because the combined use of the anti-A3(N) and anti-A3aux
polyclonal antibodies could clearly distinguish between the unsp-
liced and spliced hnRNP A3 isoforms (A3a and A3b, respectively),
these antibodies were applied to 2-D gels of the same type pre-
sented above in Fig. 1A. This final analysis, shown in Fig. 2B, veri-
fied the presence of the common hnRNP A3a isoform in human
and murine cells, in contrast to hnRNP A3b, which appeared almost
exclusively in the murine cells. The 2-D gel resolution of hnRNP
A3b revealed (as in Fig. 1) three major and at least two minor spots,
which were most likely the products of extensive post-transla-
tional modification (e.g., phosphorylation, methylation).
We then performed the immunodetection of hnRNP A3
isoforms (A3a and A3b) in extracts prepared from several mouse
tissues, including the kidney (Ki), spleen (Sp), lung (Lu), heart
(He), liver (Li) and brain, with the latter corresponding to the bulk
of brain tissue (cerebrum; Br) and to the cerebellum alone (Br/ce).
A similar amount of total protein extracted from the different
mouse tissues was resolved by SDS–PAGE followed by the immu-
nodetection of the antigenic proteins, as shown in Fig. 2C. With re-
spect to the hnRNP A3 species, the first observation was the major
difference in their relative enrichment in the extracted tissues. The
highest levels were observed in the spleen and lung and the lowest
(almost background levels) in the kidney and heart tissues; this
pattern is very similar to that of hnRNP A1. Moreover, the spleen,
lung, liver and kidney (the latter referring to a higher concentration
of a tissue homogenate; data not shown) as well as the brain cer-
ebellum, although not the brain (cerebrum) tissue, exhibited a
higher A3b–A3a ratio, in line with the mouse/rat cell lines shown
in panels A and B of Fig. 2. The A3b–A3a ratio was reversed only
in the case of the brain tissue (excluding cerebellum), which is
an interesting observation that agreed with the recently reported
findings upon the application of whole brain extracts [27].
To investigate the sub-cellular distribution of the hnRNP A3a
and A3b isoforms, we selected the liver and brain tissues to isolate
their corresponding nuclear and cytoplasmic extracts (as indicated
in Materials and methods). The liver represented all of the tissues
with a higher A3b–A3a ratio, while the brain tissue was employed
because it was the sole tissue that exhibited a reversed ratio of the
 C. Papadopoulou et al. / Archives of Biochemistry and Biophysics 523 (2012) 151–160 155

A mouse human mouse human B

A549

PDVC
Novikoff

PDVC
HeLa

A549

HeLa

A549
LA-4

LA-4
CMT
CMT
NEPHGE
OH-

SDS-PAGE
kDa kDa H+ H+ OH-

97 80
66
80
55
66 * 45
hnRNP A3a
55
hnRNP A3a 30
45
hnRNP A3b
WB: anti-A3(N)
30

WB: anti-A3(N) WB: anti-A3aux

*
hnRNP A3a
hnRNP A1
hnRNP A3b

β-actin
WB: anti-A3aux

C D Liver (Li) Brain (Br)

Ki Sp Lu He Li Br Br/ce C N C N

hnRNP A3a hnRNP A3a

short exp long exp

hnRNP A3a hnRNP A3a

hnRNP A3b hnRNP A3b

hnRNP A3a hnRNP A3a

hnRNP A3b hnRNP A3b

short exp long exp

hnRNP A1 hnRNP A1

β-actin α-tubulin

Fig. 2. Immunochemical characterization of hnRNP A3 isoforms in mouse and human cells. (A) Equal amounts (100 lg) of the proteins extracted from the three mouse
(CMT64/61, LA-4 and PVDC57) and the two human (HeLa and A549) cell lines were resolved by SDS–PAGE and transferred to nitrocellulose. Duplicate membranes were
incubated with either the commercial antibody targeting the unspliced hnRNP A3a variant alone [anti-A3(N)] or the new homemade affinity-purified anti-A3aux antibody
able to react with both A3a and A3b isoforms. The detection of a cross-reactive protein of roughly 75 kDa in the mouse extracts by the anti-A3aux antibody is marked (⁄). The
parallel immunodetection of hnRNP A1 and b-actin is also shown. (B) The 2-D resolution (NEPHGE/SDS–PAGE) of the hnRNP proteins isolated from the human (A549) and the
rat (Novikoff) cell extracts was performed as in Fig. 1A, followed by western blotting with either the anti-A3(N) or the anti-A3aux rabbit antibodies. The immunodetection of
the spots corresponding to the hnRNP A3a and A3b isoforms is shown. (C). Equal amounts (100 lg) of the soluble proteins obtained from mouse tissue homogenates
corresponding to the kidney (Ki), spleen (Sp), lung (Lu), heart (He), liver (Li) and two brain sub-areas, the cerebrum (Br) and cerebellum (Br/ce), were resolved by SDS–PAGE
and transferred to nitrocellulose. The immunodetection of the hnRNP A3a and A3b isoforms, hnRNP A1 and b-actin was performed by blotting with the appropriate
antibodies. (D). The nuclear (N) and cytoplasmic (C) extracts were obtained from portions of the liver (Li) and brain (Br) tissue (50 mg each), and an amount of the total
nuclear and cytoplasmic protein that corresponded to the same percentage (1/10th) of mouse tissue homogenate was resolved by SDS–PAGE. For this experiment, a much
higher concentration of cytoplasmic protein was loaded onto the gel than the nuclear protein (roughly 10-fold for the brain and 30-fold for the liver). The immunodetection of
the hnRNP A3a and A3b isoforms was performed on duplicate gels, and the immunodetection of hnRNP A1 and a-tubulin was performed by the sequential blotting of the
same gels. The 4-pointed star symbol is used to indicate the presence of a reactive protein impurity (of approximately 50 kDa) in the liver cytoplasmic extract. Either a short
or long ECL exposure of part of the gel (as indicated in panels C and D) that refers to the application of the anti-A3aux antibody is also included to enable a better resolution
between the hnRNP A3a and A3b isoforms within the mouse tissues.

isoform types, as observed in Fig. 2C. To estimate the relative
enrichment of the hnRNP A3 isoforms in the nuclear and cytoplas-
mic compartments, we based our comparison on tissue equiva-
lents. For this experiment, the same percentages of the extracted
nuclear (N) and cytoplasmic (C) total proteins were resolved on
duplicate gels. Western blotting was then performed to identify
hnRNP A3a and A3b in parallel with hnRNP A1 and a-tubulin
(the latter served as a cytoplasmic index protein). The results of
this analysis are presented in Fig. 2D. As clearly observed, hnRNP
A3a and A3b were highly enriched in the nuclear compartment
in both the liver and brain tissues, with a minor fraction of both
isoforms also detected in the cytoplasmic fraction, similar to the
cellular distribution of hnRNP A1. Aside from the clear reproduc-
tion of the differential ratio of A3a and A3b between the liver
and brain (also in both cellular compartments), there were no
other substantial differences observed with respect to their sub-
cellular distributions.
The present study provides the first comparison of the hnRNP
A3 variants within different mouse tissues, which led to the iden-
tification of the brain as the sole tissue thus far to contain a larger
amount of A3a than A3b.
Presence of hnRNP A3 within immunoselected hnRNP/mRNP
complexes from mammalian cell extracts
In comparison to hnRNP A1 and A2/B1, hnRNP A3 remains the
least studied member of the hnRNP A/B family in human cells. In
Fig. 3A, the immunodetection of the human hnRNP A/B is observed
156 C. Papadopoulou et al. / Archives of Biochemistry and Biophysics 523 (2012) 151–160

in the nuclear (N) and cytoplasmic (C) fractions from the HeLa and
A549 cell lines in parallel with the exclusively nuclear hnRNP C1/
C2 [35], shown for comparison, and with b-actin. The hnRNP A3a
isoform unique to human cells was mainly concentrated in the nu-
clear compartment, as observed for hnRNP A1 and A2/B1. However,
similar to hnRNP A1 (but not A2/B1), a minor fraction of hnRNP
A3a was clearly detected in the cytoplasmic fractions. These results
were in good agreement with the nuclear and cytoplasmic distri-
bution of hnRNP A3a in the mouse tissues as presented in Fig. 2D.
We then examined the ability of the anti-hnRNP A3 antibodies
to immunoselect hnRNP/mRNP complexes from fractionated
human (A549) cell extracts. However, both the anti-A3(N) and
anti-A3aux polyclonal antibodies performed very poorly in immu-
noprecipitation (IP) studies (data not shown). Due to this limita-
tion, we relied solely on the reciprocal use of the anti-hnRNP A1
monoclonal antibody that had previously enabled the detection
of hnRNP A3 among the hnRNP A1-associated protein species
[30]. Fig. 3B(a) shows the anti-A1 IP assays of the A549 nuclear
and cytoplasmic fractions with or without their prior RNase A
digestion (±RNase) in parallel with mock reactions. As observed
by the protein resolution of the immunoprecipitated protein and
the subsequent western blotting with antibodies for hnRNP A3a,
A1 and C1/C2 (the latter also served as a nuclear protein index),
hnRNP A3a was clearly associated with the hnRNP A1-selected nu-
clear and cytoplasmic RNP complexes. In both cellular compart-
ments, this association was sensitive to the prior RNase A
digestion of the extracts, indicative of an RNA-dependent interac-
tion, which was similar to the nuclear hnRNP C1/C2-A1 association.
The above IP studies were expanded to include antibodies against
CBF-A, which is another hnRNP A/B type protein. This choice was

PDVC LA-4
C

IgG-co

IgG-co
α- Α1

α- Α1
A PDVC LA-4
C N C N
HeLa A549 kDa
80
WB: anti-A3(N)

C N C N 66 IgGH
55
hnRNP A1
45 hnRNP A1
hnRNP A3a
30
hnRNP A3a
WB: anti-A3aux

80
hnRNP A2/B1
66 * IgGH
55 hnRNP A3a
45 hnRNP A3a
hnRNP C1/C2
30 hnRNP A3b

β-actin 80
IgGH
66 hnRNP A3a
55 α- tubulin
hnRNP A3b
a. b.
B
Α549: Nuclear Ex. Cytoplasmic Ex.
Input

Input

mock IP α-Α1 mock IP α-Α1 HeLa Nuclear Ex.

RNase:
α-CBF-A

α-CBF-A

- + - + - - + - + -
mock IP

hnRNP A1
A1 fr.
RNase: - - +
hnRNP A3a CBF-A

hnRNP A3a
hnRNP C1/C2

a. b.
Fig. 3. Cellular distribution and detection of hnRNP A3 isoform species within hnRNP complexes immunoselected from human and murine cell extracts. (A) The nuclear (N)
and cytoplasmic (C) extracts were prepared from two human cell lines (HeLa and A549), and equal protein amounts (50 lg) were resolved by SDS–PAGE and transferred to
nitrocellulose. The immunodetection of the hnRNP A1, A3a, A2/B1 and C1/C2 proteins is shown in parallel with b-actin following blotting with the appropriate antibodies.
(Ba) The monoclonal antibody against the hnRNP A1 protein (a-A1) was employed in immunoprecipitation (IP) assays using nuclear and cytoplasmic extracts from human
(A549) cells. For the extracts used, the protein amount corresponded to the same cell equivalent (1.7  107 cells/IP) with (+) or without () prior treatment with RNase A.
Parallel mock IP reactions in the absence of the antibody were performed as negative controls. The proteins present in the immune pellets were resolved by SDS–PAGE,
transferred to nitrocellulose and blotted with antibodies against hnRNP A1, A3a and C1/C2. The input corresponds to an aliquot of the extract applied in IP (A1fr. indicates the
degradation of hnRNP A1 that occurred in this experiment). (Bb) Similar IP assays as those in Ba were performed on a HeLa nuclear preparation using an antibody against CBF-
A. The immunodetection of CBF-A and hnRNP A3a in the immune pellets is shown. (Ca) The nuclear (N) and cytoplasmic (C) extracts were prepared as in panel A using two
mouse cell lines (PDVC and LA-4), and the immunodetection of the hnRNP A3a and A3b variants followed the application of the anti-A3(N) or anti-A3aux antibodies. The
parallel immunodetection of a-tubulin, a known cytoplasmic index protein, is also shown, as is the presence of the as yet uncharacterized cross-reactive protein (⁄) initially
presented in Fig. 2A. (Cb) The nuclear extracts corresponding to the PVDC and LA-4 mouse cells were subjected to IP reactions with the application of the a-A1 antibody (as in
Ba) in parallel with a control IP (IgG-co). Following the protein resolution of the immune pellets and transfer to nitrocellulose, the presence of the immunodetected hnRNP A1
and A3a and A3b variants was indicated. IgGH refers to the heavy IgG chain.
 C. Papadopoulou et al. / Archives of Biochemistry and Biophysics 523 (2012) 151–160
based on our previous findings that showed the ability of nuclear
actin to associate with a subset of the rat liver hnRNP A/B proteins
[25]. This subset includes CBF-A as well as the minor forms of
hnRNP A2 and A3 (excluding their major variant forms). In
Fig. 3B, panel b, the outcome of the application of the anti-CBF-A
antibody to an RNase A-treated or -untreated human (A549) nucle-
ar extract is presented. The western blotting identification of CBF-A
and hnRNP A3a in the immune pellets, in direct comparison to the
mock IP, revealed the co-precipitation of hnRNP A3a with CBF-A.
Interestingly, this association was unaffected by RNase A digestion,
in clear opposition to the RNase-sensitive hnRNP A1-A3a interac-
tion shown in panel 3B (a).
A similar procedure to that applied to the human (A549) cell ex-
tracts was also performed with mouse cell extracts, and both
hnRNP A3a and A3b isoforms could be identified. The nuclear
and cytoplasmic extracts obtained from the two mouse cell lines
(PDVC and LA-4) were blotted with either anti-A3(N) or anti-
A3aux antibodies to detect the hnRNP A3a or A3b isoforms, respec-
tively (Fig. 3C(a)). Both isoforms exhibited a major nuclear
localization with a minor fraction of hnRNP A3b detected in the
cytoplasmic compartment (see detection of a-tubulin as a cyto-
plasmic index). We note in this aspect the similar distribution of
the as yet unidentified cross-reactive 75 kDa protein. Thereafter,
the application of the anti-hnRNP A1 antibody in IP assays
performed on the mouse nuclear extracts provided additional evi-
dence for the ability of both hnRNP A3 isoforms (A3a and A3b) to
associate specifically with the hnRNP A1-selected RNP complexes
in mammals (Fig. 3C(b)).
Interaction of hnRNP A3 with other protein members of hnRNP
complexes in vitro
With the notable exception of hnRNP A3, the pattern of interac-
tions operating amongst most human hnRNP proteins has been
extensively analyzed both in vivo and in vitro [36,37]. To investi-
gate the type and nature of the interactions hnRNP A3 could have
with the other hnRNP proteins in vitro, the recombinant GST-
A3aux protein was bound to agarose–glutathione beads and em-
ployed in pull-down assays using fractionated human (A549) cell
extracts. Following the incubation of the beads with either the nu-
clear or cytoplasmic extracts, the association of the endogenous
hnRNP proteins with the recombinant GST-A3aux was investi-
gated, using recombinant GST alone as a negative control. Because
hnRNPs are mainly nuclear proteins, a higher cytoplasmic (com-
pared to nuclear) protein concentration was used in the pull-down
assays, which also improved the detection of any interactions be-
tween the endogenous hnRNPs and the recombinant proteins.
The incubated beads were then extensively washed either with
the standard low buffer (150 mM NaCl) or a higher stringency
wash buffer (300 mM NaCl). The proteins that bound to the beads
were resolved by SDS–PAGE and transferred to a membrane. In
Fig. 4B, the presence of the recombinant GST-A3aux (the approxi-
mately 42 kDa intact form as well as its fragmented forms) and GST
proteins that were added in the pull-down assays can be observed
by an initial Ponceau S staining of the membrane. Subsequent wes-
tern blotting using antibodies that targeted selected hnRNPs was
used to test their ability to co-precipitate with the recombinant
proteins. In the low stringency wash (W/150), the western blot re-
vealed the specific association of GST-A3aux with the endogenous
nuclear hnRNP A/B (A3a, A1, A2) as well as with the L and M pro-
teins. The same hnRNP proteins were also identified in the cyto-
plasmic extract with the exception of A2, which was below
detection levels (see Fig. 3A). In both cellular compartments, the
association of the hnRNP A/B proteins was sensitive to the higher
stringency wash (W/300), in clear opposition to hnRNP L and M.
We note here that, similar to the auxiliary domain of hnRNP A1
157
and A2 [36], recombinant A3aux was able to make homomeric
interactions with the endogenous hnRNP A3a and to form het-
ero-complexes with the other hnRNP A/B, A1 and A2. However,
in clear opposition to hnRNP A1 [37], GST-A3aux could also associ-
ate stably with at least two more hnRNPs, the hnRNP M and L pro-
teins, in addition to the A/B proteins.
The availability of a full-length recombinant hnRNP M [34] al-
lowed us to use a pull-down assay to test whether the interaction
between GST-A3aux and hnRNP M was direct and occurred in the
absence of any endogenous proteins. The outcome of this ap-
proach, shown in Fig. 4C, provided the novel identification of the
ability of these two recombinant species (corresponding to hnRNP
A3 and M) to form a direct protein–protein interaction in vitro that
was stable under the high-salt washing of the beads.
Additional pull-down assays were performed to examine the
properties of the observed association of hnRNP A3 with the
endogenous hnRNPs. In this experiment (see Fig. 4D), GST-A3aux
or GST alone was incubated with the fractionated nuclear and cyto-
plasmic extracts (from A549 cells) with or without their prior
RNase A-digestion. After washing the beads in the low-salt buffer,
the immunodetection of the bound hnRNP protein species was per-
formed. A major finding of this approach was that, with the excep-
tion of hnRNP A1, the interactions between GST-A3aux and the
endogenous hnRNPs A3a, A2/B1, L and M in the nuclear extract ap-
peared to be insensitive to prior RNase digestion, with a notable
RNase-induced enhancement. This result was most likely due to
the release of hnRNP proteins from the RNase-treated hnRNP com-
plexes and, thus, their increased accessibility for interaction with
GST-A3aux. An overall similar pattern of interactions as that ob-
served with the nuclear extract was observed in the cytoplasmic
extract, albeit with much lower intensity and without the detec-
tion of hnRNP A2/B1. The RNA-sensitive association of hnRNP A1
with GST-A3aux, in contrast to that observed with hnRNP L, was
also observed in the HeLa nuclear extract, as presented in Fig. 4E,
in agreement with the IP experiment (Fig. 3B) that showed an
RNA-dependent association of hnRNP A3a with A1.
Based on the above findings, the ability of the auxiliary domain
of hnRNP A3 to participate in stable protein–protein interactions
with a number of hnRNP proteins in both the nuclear and cytoplas-
mic compartments was clearly demonstrated in vitro.
Discussion
One major outcome of the present study was the establishment
of the differential protein expression of hnRNP A3 between human
and murine (rat/mouse) cells. This conclusion was based on our
investigation of hnRNP A3 in a number of human and murine cell
lines (including mouse tissue lysates) that extended and verified
our initial work on this protein [23]. In the present study, the
spliced A3b isoform was verified as the major A3 form in murine
(mouse/rat) cells but was practically undetected in human cells.
In contrast, the unspliced A3a, which was the only isoform de-
tected in humans, was found to be a relatively minor hnRNP A3
isoform in mouse/rat. In addition, the examination of a number
of mouse tissues (spleen, lung, liver, kidney, and brain cerebellum)
revealed the expected enrichment of A3b relative to A3a. However,
an important finding was that mouse whole brain lysate [the main
brain tissue (cerebrum)] exhibited higher protein levels of A3a
than A3b, indicative of a tissue-specific variation of hnRNP A3
expression. Our findings support and explain the published results
on the identification of hnRNP A3 isoforms based on the use of
neuronal cells of human and murine origin [18]. Furthermore, in
a recent study [27], a detailed examination of the species-specific
variation in the splicing patterns and the protein expression of
hnRNP A/B (A1, A2/B1 and A3) in brain extracts of human and
158 C. Papadopoulou et al. / Archives of Biochemistry and Biophysics 523 (2012) 151–160

D Nuclear Ex. Cytoplasmic Ex.

A

GST-A3aux

GST-A3aux
RNA binding domain Aux. domain

Ex. input
Ex. input
RRM RRM

GST

GST
N- -C
RNase: - + - + - + - +
A3aux
hnRNP A3a

B hnRNP A1
Nuclear Ex. Cytoplasmic Ex.
GST-A3aux
GST-A3aux

hnRNP A2/B1
GST
GST

hnRNP L
Rec. input
Rec. input

Ex. input

Ex. input
W/150
W/300

W/150
W/300
W/300
W/150

W/150
W/300

kDa hnRNP M
64
Ponseau S

49
GST-A3aux
C E

GST + Rec. M
37 HeLa nuclear ex.

GST-A3aux
GST Rec. M
GST-A3aux + Rec. M - RNase + RNase

W/150

W/300
26

GST-A3aux

GST-A3aux
GST

Ex. input
hnRNP A3a kDa

GST

GST
115
hnRNP A1 82 hnRNP M

β-actin 64 M fr.
WB

hnRNP A3a
49
hnRNP A2/B1
37
hnRNP L
hnRNP A1
hnRNP M 26
WB: anti-hnRNP M
hnRNP L

Fig. 4. Interaction of the auxiliary domain of hnRNP A3 fused to GST (GST-A3aux) with endogenous hnRNP proteins in vitro. (A) Schematic diagram of the RNA binding and
auxiliary (aux) domains common to the hnRNP A/B-type proteins. (B) The nuclear and cytoplasmic extracts (corresponding to 1 and 2 mg of total protein, respectively) were
isolated from human (A549) cells and allowed to incubate with glutathione beads that had been previously bound to recombinant GST-A3aux or GST alone in an in vitro pull-
down assay. The beads were washed in a buffer of 150 or 300 mM NaCl (W/150 or W/300). The bound proteins were resolved by SDS–PAGE and transferred to nitrocellulose.
The Ponceau S staining of the entire blot is shown. The lanes labeled as Rec. input represent an aliquot of the recombinant GST-A3aux or GST proteins, and the Ex. input lanes
represent an aliquot of the extract as applied in the pull-down assays. The membrane was then western blotted to detect the presence of hnRNP A3a, A1, A2/B1, L and M as
well as b-actin using the corresponding antibodies. (C) Pull-down assays were performed by incubating the GST-untagged full-length hnRNP M with glutathione beads that
had bound to GST-A3aux or GST alone in the absence of any extract. The beads were then washed under low (W/150) or high (W/300) salt conditions. The lane labeled as Rec.
M represents an aliquot of the recombinant hnRNP M. Western blotting was performed with a monoclonal antibody specific to hnRNP M. The presence of some fragmented
recombinant M is indicated (M fr.). (D) Pull-down assays similar to those presented in panel B were performed on A549 nuclear and cytoplasmic extracts with (+) or without
() prior incubation with RNase A. The proteins eluted from the beads (washed in low salt buffer) and an aliquot of the extract (Ex. Input) were immunoblotted with
antibodies against the hnRNP proteins A3a, A1, A2/B1, L and M. (E) Presentation of a pull-down assay (as in D) performed on a HeLa nuclear extract after western blotting with
antibodies against hnRNP A3a, A1 and L.

murine origin has been reported. An interesting observation made present work]. At this stage, only some suggestions can be made
in this report is that, in rat whole brain lysate, A3a is the predom- about a presumed requirement for increased hnRNP A3a levels in
inantly expressed isoform, despite the fact that the A3a transcript rat/mouse brain function as well as in humans. hnRNP A/B pro-
is the minor alternatively spliced form [27]. In this context, our teins, with their modular 2xRBD-Gly structure, participate in a
present analysis employing a number of mouse tissues revealed range of nucleic acid (RNA and ssDNA) and protein–protein inter-
the tissue-specific protein expression of the hnRNP A3a and A3b actions that provide for their broad range of nuclear and cytoplas-
isoforms (shown in Fig. 2C), which deserves special attention. Thus, mic functions in mRNA maturation [8,9]. The presence or absence
in addition to species-specific variations, we also uncovered the of an alternate exon in an A/B isoform is expected to modify its par-
novel tissue-specific expression of hnRNP A3 isoforms in murine ticipation in the above interactions. In the Introduction, we noted
cells. the ability of hnRNP A2/B1 (in particular, its minor A2b isoform)
The fact that brain (cerebrum) is, thus far, the only murine and A3, but not A1, to act as mRNA trafficking trans-acting factors
tissue found to overexpress the unspliced hnRNP A3a variant is ex- in both oligodendrocytes and neurons [18,19]. In addition, it has
pected to be of biological significance and requires further investi- been proposed that the inclusion of the 22-nucleotide-long N-ter-
gation. This finding should be considered along with the observed minal exon 1b that is next to the start codon in hnRNP A3a is ex-
lack of hnRNP A3b in human cells as well as the fact that such ma- pected to affect translational efficiency [27]. In the case of the rat
jor species-specific and tissue-specific variations have not been re- brain, the application of affinity pull-down assays using A2RE
ported for the other hnRNP A/B (A1 and A2/B1) members [27], RNA and telomere-specific DNA sequences resulted in the
 C. Papadopoulou et al. / Archives of Biochemistry and Biophysics 523 (2012) 151–160
over-representation of A3b as the selected variant, which was in
contrast to the overall enrichment of A3a over A3b in the brain ex-
tract [27]. It may be inferred from the above findings that the unsp-
liced hnRNP A3a isoform is not likely to play a major role in the
trafficking and localization of specific brain mRNAs or in telomere
formation. This isoform might, rather, have functions related to the
species- and tissue-specific requirements for nuclear events
(mainly splicing) as well as in the stability and translation of
mRNAs in the cytoplasm.
The difference in the overall amounts of hnRNP A3 between hu-
man and murine cells should also be of functional significance. As
clearly presented in our studies [23,26 and present work], A3b was
a major A/B type protein in murine cells, almost as abundant as
hnRNP A2/B1, but it was undetected in human cells. We believe
that the apparent functional redundancy of the hnRNP A/B proteins
A1, A2 and A3 [18,28] should compensate for this great imbalance
in the cellular concentration of hnRNP A3, an issue worth consider-
ing in future studies.
In our work, we have not examined the anticipated involvement
of hnRNP A3 in protein–RNA interactions that should be mainly
based on its N-terminal RNA-binding domain. We have only indi-
rectly addressed this issue in our IP studies by the detection of
the RNA-labile association of hnRNP A3 with the immunoselected
hnRNP A1-RNP and mRNP complexes from both human and mouse
cell extracts (Fig. 3), which indicated its direct or indirect binding
to precursor and mature mRNA. The bulk of hnRNP A3 was found
in the nucleus with a minor fraction detected in the cytoplasmic
compartment, suggesting participation in the cytoplasmic as well
as in the nuclear events of mRNA metabolism.
A limited number of studies is available concerning the protein–
protein interactions of hnRNP A3, in contrast to the other major
hnRNP A/B proteins, A1 and A2, which have been studied both
in vivo and in vitro [36,37]. The existence of RNA-independent
homomeric and heteromeric interactions between hnRNP A1 and
A2 that are dependent on the presence of the Gly-rich auxiliary do-
main of the protein has been clearly established. We presented
here for the first time in vitro studies that investigate the contact
of the Gly-rich auxiliary domain of hnRNP A3 with the other com-
ponents of the hnRNP and mRNP particles within the nuclear and
cytoplasmic extracts of human (A549, HeLa) cells. Based on the
findings shown in Fig. 4, the auxiliary domain of hnRNP A3, as in
the case of hnRNP A1 and A2, could also form homo-complexes
as well as hetero-complexes with hnRNP A1 and A2. However, in
clear opposition to the findings with hnRNP A1 and C [37], GST-
A3aux could also associate with other than A/B-type proteins, as
observed by its stable and RNA-independent association with
hnRNP M and L. It is interesting to note the persistence of this nu-
clear association in the cytoplasmic fraction (Fig. 4). As the forma-
tion of homo- and hetero-complexes (with the exception of A1)
was greatly enhanced in the RNase-treated extracts, compared to
the untreated extracts and did not require the presence of the
RNA-binding domain of the recombinant A3 protein, these com-
plexes are considered to require mainly protein–protein interac-
tions. Although RNase digestion largely removed all detectable
RNA from the extract (data not shown), we cannot exclude the pos-
sibility that a minor fraction of protected oligoribonucleotides re-
mained bound to the associated proteins and contributed to this
interaction.
The ability of the auxiliary domain of hnRNP A3 to make stable
associations with hnRNP M and L represents a feature that is thus
far unique to an A/B type protein and suggests the involvement of
hnRNP A3 in an extensive network of protein–protein interactions
within the hnRNP and, likely, mRNP complexes. As this network
could facilitate the bridging within the hnRNP particles of the core
proteins (A/B and C) with the less stably associated hnRNP proteins
(hnRNP D–U), a possibility worth considering is the contribution of
159
hnRNP A3 to the assembly and stability of hnRNP complexes in
mammals. We also wish to refer in this context to our recent study
employing (similar to human) pull-down assays on the extracts of
mouse embryonic fibroblast cells that revealed the ability of GST-
A3aux to make an RNA-independent interaction with HuR [30].
This finding extended the repertoire of feasible hnRNP A3 interac-
tions and is of relevance to the current work because HuR is an-
other RNA-binding protein known to function in the stability/
translation of mRNAs containing 30 -UTR ARE elements [33 for Refs.
therein]. HuR has also been reported to extensively associate with
the hnRNP proteins in immunoselected hnRNP and mRNP com-
plexes [30].
The broad participation of hnRNP A3 in many cellular functions
either by itself or in conjunction with the other A/B type proteins is
currently supported by a number of recent studies. Of special inter-
est is its contribution to age-related gene expression based on the
identification of hnRNP A3 as the major mouse liver protein that
binds to age-related elements in the 30 UTR of both the human
and mouse factor IX (FIX) mRNA and regulates their expression
[38]. The increase of the nuclear hnRNP A3 protein level (but not
its mRNA) with age and the dependence on its phosphorylation
status has also been reported [38]. hnRNP A3 (together with the
D-like and K) is among the small group of growth hormone-regu-
lated proteins in rat liver, thus affecting sexual dimorphism [39].
Additionally, the anti-proliferative action of the glucocorticoid hor-
mones in rat glioma cells has been shown to be related to the
abundance of a few proteins, including hnRNP A3 [40]. The latter
is an apparent contradiction to the increase of hnRNP A3 protein
levels, in concert with those of A1 and A2/B1, in human lung cancer
biopsies that we reported [41], indicative of the operation of com-
plex regulatory mechanisms involving hnRNP A3.
In summary, our findings on the novel characteristics of hnRNP
A3, along with its recently reported broad participation in mRNA
maturation and many other cellular processes, underscore the role
of this protein as an important constituent of hnRNP/mRNP com-
plexes in mammals.
Acknowledgments
This work was supported by a research Grant (PENED) co-fi-
nanced by E.U.-European Social Fund (75%) and the Greek Ministry
of Development-GSRT (25%), to A.G.
We thank Dr. Piergiorgio Percipalle (Dept. of Cell and Molecular
Biology, Karolinska Institute, Stockholm) for providing the anti-
CBF-A rabbit antibody.
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