Báo cáo khoa học: The 3¢-UTR of the mRNA coding for the major protein kinase C substrate MARCKS contains a novel CU-rich element interacting with the mRNA stabilizing factors HuD and HuR ppt

The 3¢-UTR of the mRNA coding for the major protein kinase C
substrate MARCKS contains a novel CU-rich element interacting
with the mRNA stabilizing factors HuD and HuR
Georg Wein
1
, Marek Ro¨ ssler
1
, Roland Klug
1
and Thomas Herget
1,2
1
Laboratory of Molecular Neurobiology, Institute of Physiological Chemistry and Pathobiochemistry,
Johannes Gutenberg-University, Mainz, Germany;
2
Axxima Pharmaceuticals AG, Martinsried, Germany
The expression of the major protein kinase C substrate
MARCKS (myristoylated alanine-rich C kinase substrate) is
controlled by the stability of its mRNA. While the
MARCKS mRNA is long living in quiescent fibroblasts
(t
1/2
¼ 14 h), its half-life time is drastically reduced
(t
1/2
¼ 2 h) in cells treated with phorbol esters to activate
protein kinase C (PKC) or treated with growth factors. In a
first step to study the underlying mechanism we identified
both a cis-element on the MARCKS mRNA and the cor-
responding trans-acting factors. Fusing the complete
3¢-UTR or specific regions of the 3¢-UTR of the MARCKS

gene to a luciferase reporter gene caused a drastic decrease in
luciferase expression to as low as 5–10% of controls. This
down-regulation was a result of destabilization of the chi-
meric transcript as shown by RNA run-off and Northern
blot-assays. By RNase/EMSA and UV-cross-linking
experiments, we identified a stretch of 52 nucleotides
[(CUUU)
11
(U)
8
]inthe3¢-UTR of the MARCKS mRNA
specifically recognized by two RNA-binding proteins, HuD
and HuR. These trans-acting factors are members of the
ELAV gene family and bind the MARCKS CU-rich
sequence with high affinity. Overexpression of HuD and
HuR in murine fibroblasts caused a striking stabilization of
the endogenous MARCKS mRNA even under conditions
when the MARCKS mRNA is normally actively degraded,
i.e. after treating cells with phorbol ester.
These data imply, that the identified CU-rich cis-element
of the MARCKS 3¢-UTR is involved in conferring insta-
bility to mRNAs and that members of the ELAV gene
family oppose this effect. Based on its structural and func-
tional properties, the (CUUU)
11
(U)
8
sequence described
here can be grouped into class III of AU-rich elements.
Keywords: RNA stability; AU-rich elements; protein kinase

C; MARCKS; Hu-proteins.
Expression of many genes that control cellular proliferation
and differentiation is, at least in part, adjusted by regulation
of the stability of their transcripts (reviewed in [1–3]). Such
transcripts include proto-oncogenes such as c-myc,tran-
scription factors, cytokines, lymphokines, growth factors
and their receptors. The decay rates of many of these
transcripts are governed by a sequence determinant called
the AU-rich element (ARE). This cis-acting element, which
varies in length and sequence, is characterized by a high
degree of uridylate and, sometimes, adenylate residues and
often contains one or more AUUUA pentamers [4,5]
mediating transcript instability. Moreover, ARE-directed
mRNA degradation is influenced by many exogenous
factors, including phorbol esters, calcium ionophores,
cytokines and transcription inhibitors, consistent with the
possibility that AREs play a critical role in the regulation of
gene expression during cell proliferation and differentiation
[5–8]. To date, three classes of AREs have been identified
based on their presence, number of repeats of the pentamer
AUUUA, and their subsequent effects on RNA decay [9].
Many ARE-specific RNA-binding proteins have been
identified; however, the molecular mechanism by which
these proteins target mRNA for rapid degradation remains
largely to be determined. It is not yet clear whether AREs
are the actual targets for ribonucleases and/or whether
trans-acting factors lead to an increase in the rate of
deadenylation, which often is the first step in ARE-directed
mRNA decay (reviewed in [8,10,11]).
A number of trans-acting factors interacting with AREs

have been identified. Among them are the ELAV-like
proteins, also called Hu antigens, which are the mammalian
orthologues of the elav (embryonic lethal abnormal vision)
gene of Drosophila [11]. Hu proteins are also known as
autoimmune antigens in human paraneoplastic disorders
[12]. In each species of vertebrates, there are four different
Hu proteins, which are produced from distinct genes [13].
HuC, also referred to as ple21 or ElrC, and HuD, also
referred to as ElrD, are expressed specifically in neurons,
Correspondence to T. Herget, Axxima Pharmaceuticals AG,
Am Klopferspitz 19, 82152 Martinsried, Germany.
Fax: + 49 89 740 165 20, Tel.: + 49 89 740 165 30,
E-mail:
Abbreviations: ARE, AU-rich element; CDS, coding sequence;
CstF64, cleavage stimulation factor of 64 kDa; ELAV, embryonic
lethal abnormal vision; GAP-43, growth associated protein of 43 kDa;
Luc, luciferase; MARCKS, myristoylated alanine-rich C kinase
substrate; PDB, phorbol-12,13-dibutyrate; PKC, protein kinase C;
RA, all-trans retinoic acid.
(Received 10 September 2002, revised 7 November 2002,
accepted 26 November 2002)
Eur. J. Biochem. 270, 350–365 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03396.x
whereas HuB, also referred to as Hel-N1 in humans, Mel-N1
in mice or ElrB, is expressed mainly in neurons, testes and
ovaries. Another Hu protein, HuR, also referred to as HuA
or ElrA, is expressed in all tissues tested [14]. All four
members of the Hu family encode RNA-binding proteins
containing three RNA-interacting domains of the RRM
(RNA recognition motif) type [11]. The first two domains
recognize and specifically associate with a target motif, the

ARE, whereas the third domain seems to bind the mRNA
poly(A) tail [15]. It is generally accepted that Hu proteins
exert a stabilizing function on labile mRNAs [16,17]. HuD/
HuR proteins are reported to travel between the nucleus and
cytoplasm due to the presence of a nuclear-cytoplasmic
shuttling signal [16,18–20]. As Hu proteins are associated
with mRNAs, this shuttling may represent a mechanism
used to distribute bound messengers and determine the
amounts of mRNA available for translation [20].
We previously showed that the cellular level of the
mRNA coding for the myristoylated alanine-rich C kinase
substrate, called MARCKS or 80K, is cell cycle dependent.
In quiescent Swiss 3T3 and murine embryonic fibroblasts
(MEF), levels of MARCKS mRNA are high. However,
upon stimulating cells with growth factors like PDGF [21]
or activating protein kinase C (PKC) with phorbol esters
[22], the MARCKS mRNA levels were drastically reduced
within hours. Nuclear run-off experiments showed that this
down-regulation was not due to a shut-down of the
promoter of the unique MARCKS gene, but to an
enhanced degradation of the MARCKS mRNA [21,22].
An inverse correlation between expression of MARCKS
and progression through the cell cycle was also shown after
subculturing Swiss 3T3 cells in fresh medium [23]. Further-
more, overexpression of the MARCKS protein in fibro-
blasts caused slow growth rate, a low final cell density and
enhanced susceptibility towards calmodulin antagonists
[24]. Additionally, the finding that many tumor cell lines
have no or reduced MARCKS expression supports the
hypothesis that MARCKS is a tumor and/or growth

suppressor gene in some cell types [25–27]. The precise
physiological role of this widely distributed PKC substrate
has not been convincingly established yet (reviewed in
[28–30]. Phosphorylation of MARCKS appears to be
involved in controlling cell shape changes, possibly via
regulating cytoskeleton-membrane linkage [31], and/or
adjusting the level of free cellular calmodulin [24].
The present study shows that the 3¢-UTR of the
MARCKS transcript contains a sequence that confers
mRNA instability when fused to a reporter gene. In the
3¢-UTR of the MARCKS mRNA we identified an ARE-
like sequence that is specifically recognized by several
proteins including HuD and HuR. Overexpression of HuD
induces stabilization of the MARCKS mRNA under
conditions which otherwise cause its degradation.
Materials and methods
Cell lines and culture conditions
Stock cultures of Swiss 3T3 fibroblasts [32] were propagated
as described previously [33]. For experiments, 1 · 10
5
cells
were subcultered in 90-mm dishes (Falcon) with 10 mL
Dulbecco’s modified Eagle’s medium (DMEM) supplemen-
ted with 12.5% fetal bovine serum (Life-Technologies,
Eggenstein, Germany) and incubated in a humidified
atmosphere of 10% CO
2
and 90% air at 37 °C. Cells were
rendered quiescent by incubating under these conditions for
8–10 days before use.

Swiss 3T3 cells were used for stable transfection with a
chimeric luciferase-MARCKS construct and for transient
transfection with the cDNAs coding for human HuD and
HuR.
The mouse embryonic carcinoma cell line PCC7-Mz1 is a
subclone of the PCC7-S-AzaR
1
(clone 1009) cell line. Its
properties and culture conditions have been described in
detail elsewhere [34,35]. The stem cells were maintained in
tissue culture dishes in DMEM supplemented with 12.5%
fetal bovine serum (PAA Laboratories, Co
¨
lbe, Germany) at
37 °C in 90% humidified air/10% CO
2
.
Expression of chimeric luciferase
pcDNA3-luc-MARCKS 3¢-UTR (pDK1) was constructed
as follows: pGEMÒ (Promega, Mannheim, Germany)
containing the luciferase gene was digested with StuIand
HindIII, and the luc-fragment was cloned into pcDNA3
(Invitrogen, Karlsruhe, Germany) via a filled-in EcoRI site
and a HindIII site downstream of the CMV promoter,
resulting in the plasmid pLuc. The MARCKS 3¢-UTR
fragment was excised with NotIfrompBS-DC1and
ligated into NotI digested pLuc downstream of the
luciferase coding sequence. The construction of 3¢ deletions
of pDK1 was performed as described for pBS-DC1
deletion clones (see Preparation of RNA transcripts)

except with digestion of pDK1 by ApaIandXhoI. The
resulting vectors were sequenced and named according to
the lengths of the MARCKS sequence contained (pDK2–
pDK10).
Swiss 3T3 cells (4.8 · 10
4
) were stably transfected with
1.5 lg of pDK1 (MARCKS 3¢-UTR: 1287 bp), pDK2
(999 bp), pDK8 (252 bp) or pLuc(–) plasmid DNA using
Lipofectamine plus reagent (Life Technologies, Karlsruhe,
Germany) according to the manufacturer’s instructions.
Two days after transfection, selection for stably transfected
cells began by incubation with geneticin (750 lgÆmL
)1
)
(G418; Sigma, Steinheim, Germany) containing medium
which was changed every 3 days. After 3 weeks, resistant
colonies were isolated by trypsinization within a glass
cylinder (ring cloning) and cloned by the limited dilution
technique. Clones were analyzed when cultures reached
confluence.
The luciferase activities of the established cell lines were
quantified with the luciferase reporter gene assay (Roche,
Mannheim, Germany) according to the manufacturer’s
instructions using the Lumat LB 9501 luminometer (Bert-
hold, Wildbad, Germany). The relative light units (RLU)
measured over 5 s were normalized to the amount of
protein concentration of each clone. The luciferase mRNAs
were detected by Northern blotting as described in the
following paragraph.

To monitor the transcriptional activity of the transfected
pLuc and pDK1 constructs, Swiss 3T3 cells nuclear run-off
analyses were performed as described previously [22] with
the following modifications. The cell lysate prepared from
quiescent cells was washed twice in NP-40 lysis buffer (0.5%
Ó FEBS 2003 Hu-proteins control MARCKS mRNA stability (Eur. J. Biochem. 270) 351
NP-40, 10 m
M
NaCl, 3 m
M
MgCl
2
and 10 m
M
Tris/HCl
pH 7.4) prior resuspending the nuclear pellet in glycerol
storage buffer (40% glycerol, 5 m
M
MgCl
2
,0.1m
M
EDTA
and 50 m
M
Tris/HCl pH 8.3) and snap freezing in 100 lL
aliquots in liquid nitrogen.
The run-off transcription assay was performed using
[a-
32

P]UTP as described previously [36] followed by puri-
fication of the
32
P-labeled RNA using the RNeasy Total
RNA kit (Qiagen, Hilden, Germany) according to the
manufacturer’s instructions. The incorporation of
[a-
32
P]UTP into newly synthesized RNA was determined
by Cerenkov counting, and equal amounts of radioactivity
( 10
6
c.p.m.ÆmL
)1
) were incubated with filter-immobilized
plasmids as described for Northern blotting. The target
plasmids (5 lg each) used were pBluescript KSÒ, p809.1
[22], pc-myc, pTH82 containing a 1.6-kb cDNA fragment
from the coding region of mouse cytochrome c oxidase
subunit 1 [35] and pGEMÒ with the luciferase gene. Before
binding to membrane (Hybond N; Amersham-Pharmacia,
Freiburg, Germany) plasmids were linearized by digestion
with either StuI(pGem)orEcoRI, denatured by incubation
with 0.2
M
NaOH for 5 min at room temperature, 10 min at
65 °C and 5 min on ice, and neutralized by addition of
6 · NaCl/Cit/1.4
M
Tris/HCl pH 3.5. After hybridization,

the filters were washed three times with 2 · NaCl/Cit for
5 min at room temperature followed by incubation with
2 · NaCl/Cit supplemented with 10 lgÆmL
)1
RNase A at
37 °C for additional 30 min. Subsequently, the membranes
were washed twice with 2 · NaCl/Cit, 0.2% SDS for
20 min at 50 °C,andthentwicewith0.2· NaCl/Cit, 0.2%
SDS at 65 °C for 20 min prior to audioradiography with
Kodak X-OMAT AR X-ray film.
Northern blot analysis
Cultures were washed twice with ice-cold NaCl/P
i
and
cellular RNA was extracted using the RNeasy Total RNA
kit (Qiagen). Five micrograms of total RNA per lane were
separated in 1.2% (w/v) agarose/2.2
M
formaldehyde gels
and transferred onto Hybond
TM
-N nylon membrane (Amer-
sham-Pharmacia) by capillary blot with 10 · NaCl/Cit.
Prehybridization was performed in 6 · NaCl/Cit containing
5 · Denhardt’s solution, 0.5% (w/v) SDS, 250 lgÆmL
)1
denatured salmon sperm DNA and 50% (v/v) formamide at
42 °C for 4 h. For MARCKS detection, the EcoRI insert of
clone p809.1 [22] containing 1.2 kb of the MARCKS cDNA
was gel-purified and labeled by random-priming in the

presence of [a-
32
P]dCTP [37]. The luciferase probe was a
radioactively labeled 1.7 kb HindIII/NotIfragmentofthe
pLuc plasmid. The
32
P-labeled probes were added to freshly
prepared prehybridization solution at 10
6
c.p.m.ÆmL
)1
and
incubated with the membranes overnight at 42 °C. Filters
were washed three times with 2 · NaCl/Cit, 0.2% (w/v) SDS
at 42 °C for 15 min each, followed by three times with
0.2 · NaCl/Cit, 0.2% (w/v) SDS at 60 °C for 15 min each.
The blots were exposed overnight at )80 °CtoKodak
X-OMAT AR films with intensifier screens.
Western blot analysis
For Western blotting 20 lg protein per lane were separated
by electrophoresis in 10 and 12.5% (w/v) SDS-polyacryl-
amide gels and transferred onto poly(vinylidene difluoride)
membranes (PVDF; Immobilon P, Millipore, Eschborn,
Germany) by semidry blotting. The membranes were
blocked with NaCl/P
i
/Triton [0.1% (w/v) Triton X-100 in
NaCl/P
i
, pH 7.2], supplemented with 5% (w/v) low fat milk

powder, for 1 h at room temperature and then incubated
overnight at 4 °C with the antibody. The Hu antiserum was
diluted 1 : 1000 in NaCl/P
i
/Triton containing 1% (w/v) low
fat milk powder, the anti-CstF64 Ig (murine mAb 3A7,
kindly provided by I. Mattaj, EMBL Heidelberg) was
diluted 1 : 100. After five washes with the NaCl/P
i
/Triton,
the membranes were incubated with horseradish peroxi-
dase-conjugated goat-anti rabbit Ig for Hu detection and
with horseradish peroxidase-conjugated rabbit-anti mouse
Ig for CstF64 detection, respectively, for 1–2 h at room
temperature. Both secondary antibodies (Dako, Hamburg,
Germany) were diluted 1 : 2000 in NaCl/P
i
/Tritonwith1%
(w/v) low fat milk powder. Membranes were washed five
times in NaCl/P
i
/Triton and bound antibodies were visu-
alized by enhanced chemiluminescence (ECL) detection
system using Fuji medical X-ray films.
Preparation of RNA transcripts
To clone the complete MARCKS 3¢-UTR sequence by
PCR we used oligonucleotides as DNA primers whose
sequences were deduced from the murine MARCKS cDNA
[38]. The PCC7-MzN1 NM 1149–cDNA library [39] was
utilized as template and the 1287 bp PCR fragment was

cloned into the EcoRI and HindIII site of pBluescript
(Stratagene, Amsterdam, Netherlands). Sequence compari-
son of the resulting plasmid pBS-MARCKS 3¢-UTR (pBS-
DC1) revealed identity with the published MARCKS
sequence [22,38].
pBluescript plasmids containing 3¢ truncated MARCKS
3¢-UTR sequences were constructed by exonucleolytic
digestion [40,41]. Following digestion with KpnIandXhoI
pBS-MARCKS 3¢-UTR was treated with exonuclease III
(100 UÆlg
)1
DNA) at 37 °C for various periods of time
(1.5–15 min). The overhanging 5¢-and3¢-ends were blunted
by incubation with nuclease S1 (20 U) for 30 min at 30 °C.
Finally, the reaction was stopped by adding EDTA (final
concentration 60 m
M
) and Tris/HCl pH 8.0 (final concen-
tration 0.3
M
). The DNA fragments were gel-purified, auto-
ligated, and transformed in E. coli C600 cells. The precise 3¢
ends of the MARCKS 3¢-UTR deletion clones were
determined by sequencing and the deletion clones named
according to their lengths, i.e. pBS-DC2 contained a 1097-
bp insert and pBS-DC10 a 75-bp insert.
The pBS-MARCKS 52 nt CU-element plasmid (pBS-
MARCKS 52 nt) was constructed by annealing the two
synthetic oligonucleotides (sense: 5¢-CCC CGG GCC CGA
ATT CCT TTC TTT CTT TCT TTC TTT CTT TCT TTC

TTT CTT TCT TTC TTT TTT TTT TTC TCG AGC
CCC-3¢;antisense:5¢-GGG GCT CGA GAA AAA AAA
AAAGAAAGAAAGAAAGAAAGAAAGAAA
GAA AGA AAG AAA GAA AGG AAT TCG GGC
CCG GGG-3¢) representing base pairs 1830–1881 of the
MARCKS cDNA [38] and cloning the resulting double-
stranded DNA into the SmaI site of pBluescript.
For in vitro transcription pBS-DC1 was linearized
with HindIII and the deletion clones with PvuII. The
352 G. Wein et al. (Eur. J. Biochem. 270) Ó FEBS 2003
pBS-MARCKS 52 nt plasmid was digested with BamHI
when in vitro transcription (T3 polymerase) of RNA in sense
orientation and with EcoRV when transcription (T7
polymerase) of antisense RNA was performed. The same
restriction sites and RNA polymerases were applied when
the empty pBluescript (pBS) vector was used for production
of negative control RNAs. All templates were phenol/
chloroform extracted and ethanol precipitated before use.
The reaction mix contained 6 m
M
MgCl
2
,2 m
M
spermidine,
10 m
M
dithiothreitol, 40 m
M
Tris/HCl pH 8.0, 0.75 m

M
each of ATP, GTP and CTP, 30 l
M
UTP, 50 lCi
[a-
32
P]UTP (3000 CiÆmmol
)1
,ICN,Eschwege,Germany),
40 U RNasin (MBI-Fermentas, St Leon-Roth, Germany),
1 lg of template DNA and 20 U of T7 or T3 polymerase
(Roche, Mannheim, Germany). After 2 h at 37 °Cthe
reaction was terminated by digestion of template DNA with
40 U DNase I (Roche, Mannheim, Germany) for further
20 min. Following removal of unincorporated nucleotides
via Sephadex G-75 (Amersham-Pharmacia, Freiburg,
Germany) gel filtration, the RNA was phenol/chloroform
extracted and ethanol precipitated. The nonlabeled com-
petitor transcripts were synthesized under the same condi-
tions, except the concentration of all four ribonucleotides
was 0.75 m
M
and the [a-
32
P]UTP was omitted.
Production of recombinant Hu proteins
To acquire recombinant GST-HuD fusion protein we
amplified a cDNA encoding residues 2–373 of HuD by
PCR using clone kuniZAP-265114 as template and BamHI
and SmaI sites containing primers (sense: 5¢-TAG CGG

ATC CGA GCC TCA GGT GTC AAA TGG-3¢;
antisense: 5¢-AAT GCC CGG GTC AGG ACT TGT
GGG CTT TGT-3¢). The plasmid kuniZAP-265114, kindly
provided by M. Kock, BASF, Germany, bears the complete
HuD coding sequence and 3¢-UTR. The resulting PCR
product (1139 bp) was cloned in frame via the BamHI and
SmaI sites downstream of the GST (glutathionine
S-transferase) gene into GEX-2T (Amersham-Pharmacia)
and was called pGEX-HuD. pGEX-HuR was constructed
in a similar way and was a gift of H. Kleinert, Mainz,
Germany. Both vectors (pGEX-HuD/pGEX-HuR) were
transformed in E. coli. (XL-1 Blue, Stratagene) and the
expressed GST-HuD/GST-HuR fusion proteins were puri-
fied exactly as described for GST-MARCKS [42].
To produce His-tagged HuD fusion protein, the HuD
PCR-fragment was digested with BamHI and SmaIasfor
GST-HuD and cloned into pQE30 (Qiagen) digested with
the same enzymes, resulting in plasmid pQE30-HuD. The
His
6
-tagged HuD fusion protein was expressed in E. coli.
(XL-1 Blue) and purified as described for His
6
-MARCKS
[43].
The protein concentrations of GST-HuD, GST-HuR and
His
6
-HuD were determined by loading aliquots on 10%
SDS/polyacrylamide gels and comparison with defined

amounts of BSA standards after Coomassie Blue staining.
Gel retardation assay
The interaction of recombinant Hu-proteins with RNA
transcripts were analyzed by gel retardation assays des-
cribed by Chung and coworkers [44]. Approximately
3000 c.p.m. (2.5 ng) of labeled RNA was incubated with
protein in a buffer containing 150 m
M
NaCl, 0.25 mgÆmL
)1
tRNA, 0.25 mgÆmL
)1
BSA and 50 m
M
Tris pH 7.0 in a
final volume of 20 lL. The reaction mixture was incubated
at 37 °C for 10 min and then 4 lL of a dye mixture [50%
(v/-v) glycerol, 0.1% (w/v) bromophenol blue and 0.1%
(w/v) xylene cyanol] were added. Twenty-five percent of the
sample (6 lL) were immediately loaded on a 0.8% (w/v)
agarose gel in Tris/acetate/EDTA buffer (1 m
M
EDTA,
40 m
M
Tris acetate pH 7.0) and gel electrophoresis was
carried out at 40 V for 2–3 h. Finally, the gels were dried
under vacuum and exposed to Kodak X-OMAT AR films
at )80 °C.
Generation of rabbit anti-Hu serum

The full length recombinant GST-HuD protein was used for
immunization of a rabbit as described for MARCKS [42]
and GAP-43 [45]. The serum was affinity-purified using the
recombinant His
6
-HuD fusion protein coupled to Affi-Gel
10 matrix (Bio-Rad, Mu
¨
nchen, Germany) as described
[45,46].
Preparation of nuclear and cytosolic cell extracts
For total cytoplasmic extracts quiescent or PDB (phorbol-
12,13-dibutyrate) (Sigma) treated cells (5 h, 200 n
M
)were
rinsed twice with ice-cold NaCl/P
i
andscrapedoffthedish
with a rubber policeman in 100 lL lysis buffer [20 m
M
KOAc, 50 m
M
MgCl
2
,2m
M
dithiothreitol, 0.5% (v/v)
Nonidet P-40, 30 m
M
Tris/HCl pH 7.1, 0.1 m

M
Na
3
VO
4
,
10 m
M
NaF, 20 m
M
2-glycerophosphate, 400 n
M
okadaic
acid, 100 lgÆmL
)1
leupeptin, 100 lgÆmL
)1
aprotinin,
10 m
M
benzamidine and 2 m
M
phenylmetylsulfonyl fluor-
ide]. Following 10 min of incubation on ice for complete
lysis the homogenate was centrifuged for 10 min at
10.000 r.p.m. (centrifuge 5417R, Eppendorf, Hamburg,
Germany) at 4 °C and the supernatants were removed
andstoredat)20 °C.
For fractionation into nuclear and cytosolic extracts the
cells were rinsed once with ice-cold NaCl/P

i
,scrapedoffthe
dish in 1 mL NaCl/P
i
and centrifuged for 5 min at
2000 r.p.m. (Megafuge 1.0R, Heraeus, Hanau, Germany)
and 4 °C. The pellet was resuspended in 100 lL of buffer A
(1.5 m
M
MgCl
2
,10m
M
KCl, 1 m
M
dithiothreitol, 10 m
M
Hepes pH 7.9, 0.1 m
M
Na
3
VO
4
,10m
M
NaF, 20 m
M
2-glycerophosphate, 400 n
M
okadaic acid, 100 lgÆmL

)1
leupeptin, 100 lgÆmL
)1
aprotinin, 10 m
M
benzamidine
and 2 m
M
phenylmethylsulfonyl fluoride) and swelled on
ice for 15 min. The cells were homogenized by pressing
through a narrow-gauge hypodermic needle and the extract
was centrifuged for 5 min with 14 000 r.p.m. at 4 °C
(Eppendorf centrifuge 5417R). The supernatant containing
the cytosolic fraction was collected and kept on ice. The
nuclear pellet was resuspended in 100 lL of buffer C
(1.5 m
M
MgCl
2
,0.2m
M
EDTA, 25% glycerol, 0.42
M
NaCl, 1 m
M
dithiothreitol, 20 m
M
Hepes pH 7.9, 0.1 m
M
Na

3
VO
4
,10m
M
NaF, 20 m
M
2-glycerophosphate, 400 n
M
okadaic acid, 100 lgÆmL
)1
leupeptin, 100 lgÆmL
)1
aproti-
nin, 10 m
M
benzamidine and 2 m
M
phenylmethylsulfonyl
fluoride), extracted for 30 min on ice and centrifuged with
14 000 r.p.m. for 15 min at 4 °C in parallel with the
Ó FEBS 2003 Hu-proteins control MARCKS mRNA stability (Eur. J. Biochem. 270) 353
cytosolic fractions. The supernatants were collected and
stored at )80 °C. The protein concentrations of all extracts
were determined using the BCA reagent (Pierce, Bonn,
Germany).
RNase/EMSA and UV-crosslinking
For RNase/EMSA analysis, between 1 and 20 lgprotein
were incubated with
32

P-labeled transcript (2.5 ng in vitro
transcribed RNA,  3000 c.p.m.) in 30 lLof50m
M
KCl,
5% (v/v) glycerol, 0.1% (v/v) Nonidet P-40, 1 m
M
MgCl
2
1m
M
dithiothreitol, 10 m
M
Tris/HCl pH 8.0 and 10 lg
yeast tRNA for 20 min at room temperature. For compe-
tition studies the nonlabeled competitor RNAs were
preincubated with the proteins for 10 min before the
32
P-labeled transcript was added. In supershift experiments
NP-40 was omitted from the buffer and the proteins were
preincubated with 4 lLofthea-Hu and the a-CstF64
antibodies for 30 min on ice. To eliminate non-protein
covered RNA sequences, 10 U of RNase T1 (Roche,
Mannheim, Germany) were added and incubation contin-
ued for an additional 30 min. Samples were then subjected
to electrophoresis (2 h at 200 V; 4 °C) performed on a 5%
(w/v) native polyacrylamide gel (37 : 1) using Tris/borate/
EDTA as electrophoresis buffer. Gels were dried under
vacuum and exposed to Kodak X-OMAT AR films at
)80 °C for 1–3 days.
For UV crosslinking assays, 5–20 lg cell extract or

different amounts of recombinant Hu-proteins were incuba-
ted with 2.5 ng radiolabeled transcript in the same buffer as
described above for RNase/EMSA analysis. After 20 min
samples were placed on ice and irradiated by 180 mJ UV
light with a Stratalinker UV 1800 (Stratagene). Then the
covalently linked RNA:protein complexes were treated with
10 lg RNase A (Roche) for 30 min at room temperature.
Reaction was stopped by addition of 30 lL2 · SDS-sample
buffer and heating for 10 min at 95 °C. The samples were
loaded on a 12.5% (w/v) SDS-polyacrylamide gel and
separated overnight with 50 V at room temperature. Finally,
the gel was dried and analyzed directly by autoradiography.
Transient expression of HuD and HuR
For transient expression of HuD and HuR in Swiss 3T3
cultures, we employed the plasmids pTetoff and pTRE
(expression vector with the tTA-regulated promoter) of the
Tetoff
TM
system (Clontech, Heidelberg, Germany) [47]. For
construction of pTRE-HuD the plasmid kuniZAP-265114
was digested with XhoI, filled-in with DNA polymerase I,
and subsequently restricted with SacII. The purified frag-
ment (1524 bp) was ligated into SacII and XbaI-blunted sites
of pTRE. Finally, the authenticity of the obtained plasmid
was verified by restriction site mapping and sequencing. The
cDNA of HuR was amplified using plasmid pZeoSV(–)HuR
sense (kindly provided by A. Levy, Technion, Institute of
Technology, Haifa, Israel) as template. The HuR-PCR
fragment (1133 bp) was cloned using EcoRI (upstream) and
XbaI (downstream) restriction sites into the pTRE vector.

Swiss 3T3 were plated the day before transfection at a
density of 2.9 · 10
4
cellsÆcm
)2
in 90-mm dishes. Cells were
transfected with either pTRE-HuD or pTRE-HuR in the
presence of plasmid pTetoff with lipofectamine 2000 (Life
Technologies, Karlsruhe, Germany) according to manufac-
turer’s instructions. After 24 h, half of the cultures were
treatedwith200n
M
PDB (5 h). Cells were harvested and
analyzed by Northern blotting as described above.
Results
MARCKS 3¢-UTR mediates mRNA instability
of luciferase reporter gene
We previously showed that elevated levels of MARCKS
mRNA and protein in quiescent Swiss 3T3 cells drastically
decreases when cultures are treated with activators of PKC
(e.g. PDB or growth factors) or when cells are plated at low
density in fresh medium [21–23]. This pronounced down-
regulation of MARCKS was caused by post-transcriptional
mechanisms involving destabilization of the MARCKS
mRNA [21,22]. As the stability of most mRNAs has been
shown to be regulated by sequences in their 3¢-UTR [3,6,48]
we explored whether the MARCKS 3¢-UTR is involved in
controlling mRNA stability. We fused the complete
MARCKS 3¢-UTR cDNA with a luciferase reporter gene
cloned into the pcDNA3 vector and stably transfected

Swiss 3T3 cells with this construct (pDK1) (Fig. 1A). In the
same way we transfected the constructs pDK2 and pDK8
containing truncated sequences of MARCKS 3¢-UTR or as
a control the luciferase reporter gene without additional
sequences (pLuc) into Swiss 3T3 cells (Fig. 1A). Cell
extracts of the established cell lines were prepared, luciferase
activities measured, normalized to protein concentration of
each clone and the average of each construct presented as
RLU (Fig. 1B). The activity of the reporter gene decreased
dramatically with the length of the fused MARCKS
3¢-UTR sequences. Swiss 3T3 cells transfected with pDK8
or pLuc bearing only limited or no MARCKS sequences
(pDK8, pLuc) showed high luciferase activities (mean:
pDK8, 2003 RLUÆlgprotein
)1
, n ¼ 6; pLuc, 2642 RLUÆlg
protein
)1
, n ¼ 7). Significantly less luciferase activity was
detected when pDK1 (complete MARCKS 3¢-UTR) or
pDK2 (MARCKS 3¢-UTR missing only the last 288
nucleotides) were transfected (mean: pDK1, 53 RLUÆlg
protein
)1
, n ¼ 15; pDK2, 184 RLUÆlgprotein
)1
, n ¼ 21).
To investigate whether the low luciferase activities were due
to reduced mRNA levels we performed Northern blot
analyses (Fig. 1C). Total RNA of four randomly chosen

clones transfected with the various luciferase constructs and,
as control, mock (pcDNA3) transfected Swiss 3T3 cells was
isolated and hybridized with a radioactively labeled luci-
ferase cDNA probe. The detected luciferase mRNA band
migrated accordingly to the length of the fused MARCKS
3¢-UTR sequences (Fig. 1A). In pDK1- and pDK2-trans-
fected cells the luciferase mRNA was hardly evident,
however, considerable signals were obtained in pDK8 and
pLuc transfectants (Fig. 1C). Thus, the amount of luciferase
mRNA of each clone corresponded closely with the level of
luciferase activity (Fig. 1B). Discrepancies may be due to
clonal selection of the transfected cell line investigated for
RNA expression. As expected, in mock transfected
Swiss 3T3 cells (3T3) no luciferase-mRNA was detected.
For monitoring the rate of transcription in pLuc- and
pDK1-transfected cells we performed nuclear run-off
assays. The nuclei of pLuc- and pDK1-transfected
354 G. Wein et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Swiss 3T3 cells were isolated and after incubation with
[a-
32
P]UTP the radioactively labeled transcripts were
hybridized to filter-immobilized cDNAs of luciferase,
MARCKS (coding region), c-myc and cytochrome c oxi-
dase. As negative control pBluescript vector DNA was
spotted on the filter. The CMV promoter activity driving the
luciferase gene was similar in pDK1- and pLuc-transfect-
ants (Fig. 1D). Transcription rate of the control mRNAs
(MARCKS, c-myc, cytochrome c oxidase) were similar in
both cell lines.

Taken together, these data reflecting the transcriptional
activity as well as the RNA- and protein levels demonstrate
that MARCKS 3¢-UTR sequences of pDK1 and pDK2
destabilize the chimeric luciferase-MARCKS mRNA via
post-transcriptional mechanisms and that the MARCKS
3¢-UTR contains a regulatory cis-element(s) mediating
mRNA instability.
A 52 nt CU-rich element is recognized by Swiss 3T3
proteins
To identify proteins binding to the MARCKS mRNA, we
cloned the complete murine MARCKS 3¢-UTR into the
pBluescript vector (pBS-DC1; Fig. 2A), synthesized radio-
actively labeled MARCKS 3¢-UTR RNA by in vitro
transcription and incubated the resulting transcript with
cytoplasmic extract of quiescent Swiss 3T3 cells. After
RNaseT1 digestion and native PAGE the formation of
two RNA:protein complexes (C1 and C2) were observed
(Fig. 2B, lane 4). Complexes were absent when the
transcript was incubated with an unrelated protein
(BSA, lane 1). To localize more precisely the site within
the MARCKS 3¢-UTR that interacts with Swiss 3T3
proteins, we performed RNase/EMSA analyses with 10 3¢
truncated MARCKS 3¢-UTR transcripts (data not
shown). Strong RNA:protein complexes were only
observed with RNA containing sequences between nucleo-
tides 1773 and 1950, i.e. with RNA derived from pBS-
DC4 (lane 5) but not with RNA from pBS-DC5 (Fig. 2B,
lane 6). Because previous work showed the importance of
U-rich sequences in mRNA stability we suspected that the
highly CU-rich sequence of 52 nucleotides on the pBS-

DC4 RNA could be involved in protein interaction
(Fig. 2A). To examine protein-binding capacity we cloned
the 52 nt sequence into pBluescript, synthesized radio-
labeled RNA and incubated the transcript with cytoplas-
mic protein of Swiss 3T3 cultures. As shown in Fig. 3A
(left panel) the formation of two RNA:protein complexes
with identical mobility to those observed with the full-
length 3¢-UTR (C1, C2) were observed with the 52 nt
RNA probe. To monitor the specificity of protein binding
to the CU-rich sequence we transcribed the pBS-
MARCKS-52 nt construct in the antisense orientation
Fig. 1. The MARCKS 3¢-UTR confers mRNA instability when fused to
the luciferase reporter gene. (A) The chimeric constructs consisting of
luciferase coding sequence (CDS) and MARCKS 3¢-UTR were driven
by a CMV-promoter and used for transfection of Swiss 3T3 fibro-
blasts. Length of the MARCKS 3¢-UTR sequences [38] of pDK1:
1310–2597 bp (complete 3¢-UTR of MARCKS); pDK2: 1310–
2309 bp; pDK8: 1310–1562 bp. The poly(A) signal of the bovine
growth hormone was provided by the pcDNA3 vector. (B) Luciferase
activity of transfected Swiss 3T3 cells were measured and the average
(± SEM) obtained with each construct is depicted. The transfectants
(1 · 10
5
cells) were seeded on 90-mm dishes and grown to confluence.
Cell extracts were prepared and luciferase activity measured using
luminometer Lumat LB 9501. Signals within 5 s were normalized to
the protein concentration of each sample and expressed as relative light
units (RLU). The lowest activity of luciferase was observed when the
complete MARCKS 3¢-UTR was fused to the luciferase gene (pDK1).
(C) The level of luciferase mRNA was determined by Northern blot

analysis. pDK1, pDK2, pDK8, pLuc and pcDNA3-transfected
Swiss 3T3 cells were seeded on 90-mm-dishes (1 · 10
5
cells). After
1 week total RNA was isolated and 5 lg per lane loaded onto a 1.2%
agarose/2.2% formaldehyde gel. After electrophoresis and transfer on
a nylon membrane, RNA was hybridized with a
32
P-radiolabeled lu-
ciferase cDNA probe (1.7 kb HindIII/NotIfragmentofpLuc)and
exposed to X-ray film (Kodak AR) with intensifier screens at )80 °C
for 4 days. The upper panel shows the autoradiography. Ethidium
bromide staining proved equal loading (lower panel). The positions of
28S and 18S rRNAs are indicated. (D) Transcriptional activity of the
luciferase gene in pLuc- and pDK1-transfected Swiss 3T3 cells were
studied by nuclear run-off assays. Plasmids containing inserts encoding
for MARCKS (p809.1), luciferase (pGEM), c-myc (pc-myc), cyto-
chrome c oxidase (pTH82) and the control vector pBluescript (KS
+
)
(pBS), were hybridized with
32
P-labeled run-off transcripts from nuclei
isolated from confluent Swiss 3T3 cultures transfected with pLuc and
pDK1. Equal amounts of radioactivity was used for hybridization. All
genes analyzed are transcribed to similar degrees in pDK1- and pLuc-
transfected cell lines. There was no hybridization signal with the pBS
vector DNA detectable. Filters were exposed to X-ray film (Kodak
AR) for 6 days at )80 °C with intensifier screens.
Ó FEBS 2003 Hu-proteins control MARCKS mRNA stability (Eur. J. Biochem. 270) 355

and incubated this RNA with extracts in the same way.
There was essentially no binding activity detected
(Fig. 3A, right panel). The sequence specificity of the
RNA:protein complexes was further demonstrated by
competition experiments (Fig. 3B) showing the complex
formation was only inhibited by the addition of increasing
amounts of unlabeled 52 nt sense transcripts (lane 4–9)
but not with 52 nt antisense RNA (lane 10–14). We
therefore conclude that the 52 nt CU-rich sequence
represents a cis-element interacting with proteins from
Swiss 3T3 cells.
By screening the MARCKS 3¢-UTR for sequences
related to the 52 nt element, we identified a series of 18 U
residues (nucleotides 1585–1603). It is likely that this
element binds, albeit more weakly, the same proteins as
the 52 nt CU element and therefore is responsible for the
faint formation of complexes C1 and C2 with construct
pDC5 (Fig. 2B, lane 6). We did not detect any protein
binding sequences within the first 200 bp of the 3¢-UTR
(nucleotides 1310–1409) of the MARCKS mRNA (data not
shown).
Detection of four proteins binding to the MARCKS
52 nt CU-rich RNA
To identify proteins responsible for the formation of the two
complexes identified in RNase/EMSA analyses, we per-
formed high resolution UV-crosslinking assays (Fig. 3C).
Five micrograms of protein of cytoplasmic Swiss 3T3
extract were incubated with radiolabeled MARCKS 52 nt
sense-RNA and subjected to UV light irradiation (180 mJ).
The crosslinked samples were treated with RNase A and

resolved by electrophoresis on a 12.5% (w/v) SDS-poly-
acrylamide gel. Four strong bands resulting from proteins
crosslinked with the radioactively labeled RNA were
detected (Fig. 3C, lane 1). The sizes of these proteins were
55, 40, 36 and 30 kDa.
Because a common feature of RNA-binding proteins is
that they can also bind single-stranded DNA [79], we
preincubated the 3T3 proteins with increasing amounts of
52 nt sense DNA-oligonucleotide. Addition of 10 ng and
more of the 52 nt sense DNA-oligo resulted in a strong
competition with the radioactive RNA for proteins and
consequently a decrease in detection of proteins crosslinked
Fig. 2. Formation of two major complexes between Swiss 3T3 proteins
and the 3¢-UTR of the MARCKS mRNA. (A) The MARCKS 3¢-UTR,
the stop codon UAA of the coding sequence (CDS) and the poly(A)
sequence are depicted. The box within the 3¢-UTR marked the iden-
tified CU-rich sequence interacting with Swiss 3T3 proteins (top). The
fragments of the 3¢-UTR cloned into plasmids (pBS-DC1, pBS-DC4,
pBS-DC5 and pBS-MARCKS-52nt) are schematically presented
(sequence according to [38]). These constructs were used to synthesize
truncated RNA segments of the MARCKS 3¢-UTR. The 3¢ termini of
pBS-DC4 and pBS-DC5 are marked with arrows and the 52-nucleo-
tide CU-rich sequence is underlined (bottom). (B) Interaction between
Swiss 3T3 proteins and the MARCKS 3¢-UTR RNA was monitored
by RNase/EMSA analysis. pBS-DC1 was digested with HindIII, pBS-
DC4 and pBS-DC5 with PvuII and used as templates for in vitro
transcription in the presence of [a-
32
P]UTP and T7-RNA polymerase.
2.5 ng of the radiolabeled RNAs were incubated for 20 min at room

temperature with 1 lg bovine serum albumin (BSA) (lanes 1–3) and
1 lg Swiss 3T3 cytoplasmic extract from quiescent cells (lanes 4–6).
Following RNase T1 digestion for 30 min at room temperature the
samples were loaded on 5% native polyacrylamide gel. After electro-
phoresis at 4 °C the gel was dried and exposed to X-ray film (Kodak
AR) with intensifier screens at )80 °C for 1 day. The positions of the
two RNA:protein complexes (C1, C2) are indicated.
356 G. Wein et al. (Eur. J. Biochem. 270) Ó FEBS 2003
with the radioactive CU-rich RNA (Fig. 3C). A similar
competition in UV crosslinking assays was observed with
unlabeled 52 nt CU-rich RNA (data not shown). Using the
corresponding antisense sequence of the 52 nt CU-rich
sequence as RNA or DNA for competition did not result in
loss of detection of the RNA:protein complexes (data not
shown). Furthermore, using the 52 nt element in antisense
orientation as probe for UV crosslinking experiments did
not reveal the four RNA:protein complexes (Fig. 3C, lane
5). Taken together, the four RNA-binding proteins identi-
fied (Fig. 3C) interact specifically with the CU-rich
sequence.
Binding of ELAV/Hu proteins to the MARCKS 52 nt
CU-rich RNA
One of the identified proteins binding to the MARCKS
CU-rich element has an apparent molecular mass of about
36 kDa (Fig. 3C). A possible candidate for this protein
might be HuR, an ubiquitously expressed, 36-kDa member
of the ELAV/Hu gene family [49]. These RNA-binding
proteins recognize U-rich sequences of RNAs coding for
proteins regulating cell growth and differentiation [49,50].
To determine if proteins of the ELAV/Hu family bind the

MARCKS 52 nt RNA, we focused on two members known
Ó FEBS 2003 Hu-proteins control MARCKS mRNA stability (Eur. J. Biochem. 270) 357
to regulate mRNA stability: the wide-spread HuR and the
neuron-specific HuD [51]. The cDNAs of human HuR and
HuD were cloned into prokaryotic expression vectors and
recombinant GST-HuD and HuR were expressed in E. coli.
Purified GST-HuD and GST-HuR fusion proteins were
incubated with radioactively labeled 52 nt sense transcripts
and HuD:RNA or HuR:RNA complex formation assayed
by gel retardation analysis [44]. The 52 nt RNA was bound
by both Hu-proteins very efficiently, and complex forma-
tion was easily detectable with HuD (0.8 n
M
)(Fig.4A)or
HuR (4 n
M
) (Fig. 4B), respectively. Using the complete
MARCKS 3¢-UTR as RNA probe complex formation with
GST-HuD and GST-HuR could be observed in the same
way (data not shown). In contrast, no specific interaction
was observed with control RNA (pBSD52ntCU-RNA) and
GST-HuD (up to 800 n
M
) (Fig. 4C). Furthermore, complex
formation was not detected with GST alone and the 52 nt
CU-rich RNA (Fig. 4D).
To explore whether the crosslinked protein of 36 kDa is
endogenous HuR (Fig. 3C), we performed an UV-cross-
linking assay with cytoplasmic extract prepared from
Swiss 3T3 cells and the radioactively labeled MARCKS

52 nt sense transcript as probe. The 36-kDa complex of
the RNA with an endogenous protein (Fig. 4E, lane 1)
was disrupted by adding increasing amounts of recom-
binant His
6
-HuD to the incubation mix (lanes 2–7). HuD
has similar binding characteristics to HuR but is not
expressed in murine fibroblasts [19] (see also Fig. 6B) and
is of different size. Due to their different sizes, the cellular
36-kDa HuR protein can easily be discriminated from the
recombinant His
6
-HuD protein (39 kDa). Addition of
His
6
-HuD apparently displaced the endogenous HuR
(p36) protein from the complex with the CU-rich element
demonstrating that both proteins are competing for
similar RNA sequences (lanes 2–7). Interestingly, adding
His
6
-HuD (5 ng and more) efficiently prevented both
binding of cellular HuR and of the unknown 55-kDa
protein to the MARCKS 52 nt RNA probe arguing that
more than one protein recognize the same site (Fig. 4E,
lanes 5–7).
Analysis of HuR and CstF64 in Swiss 3T3 cells
The data so far showed that HuR binds the CU-element
efficiently in vitro and that addition of recombinant HuD to
Swiss 3T3 extracts can compete with an endogenous

RNA:protein complex probably containing HuR. It is
known that HuR shuttles between nucleus and cytoplasm
and displays a cell-type specific subcellular distribution. To
study the identified RNA-binding proteins in more detail we
compared nuclear and cytoplasmic fractions of Swiss 3T3
cells in their capacity to bind the CU-rich sequence with the
expression pattern of HuR.
Firstly, we generated and affinity-purified a polyclonal
antiserum directed against recombinant GST-HuD fusion
protein. This Hu antiserum recognized several members of
the ELAV/Hu family due to their high degree of sequence
identity [49,51,78]. Using our Hu-specific serum and
nuclear and cytoplasmic extracts from quiescent Swiss 3T3
cells for Western blot analysis resulted in one specific band
of 36 kDa, consisting of the HuR protein (Fig. 5A). In
Swiss 3T3 cells HuR was mainly localized in the nucleus
and only a minor portion resided in the cytoplasm
(Fig. 5A, left panel) as described previously for other cell
types [16,20,51–53]. To validate the quality of our
fractionation procedure, we analyzed the samples for the
known nuclear RNA-binding protein CstF64 that recog-
nizes U-rich sequences and contributes to polyadenylation
of mRNAs [54,55]. The CstF64-specific monoclonal
antibody 3A7 [55] was used for Western blot analysis.
CstF64 was not detected in the cytoplasmic fraction, but
was exclusively in the nuclear fraction (Fig. 5A, right
panel).
Using these fractionated extracts for UV crosslinking
studies with the radiolabeled 52 nt CU-rich RNA revealed
that the intensity of the p36 RNA:protein complex (Fig. 5B)

Fig. 3. Swiss 3T3 proteins recognize the MARCKS 52 nt CU-rich
RNA element with high sequence-specificity. (A) pBS-MARCKS-52nt
was linearized with BamHI (sense) or EcoRV (antisense) and in vitro
transcribed with [a-
32
P]UTP using T3-RNA polymerase (sense) or
with [a-
32
P]ATP and T7-RNA polymerase, respectively. 2.5 ng of the
radiolabeled RNAs were incubated for 20 min at room temperature
with reaction buffer alone (lanes 1 and 4), with 1 lg bovine serum
albumin (BSA) (lanes 2 and 5), with 1 lg protein of total extract from
Swiss 3T3 cells (lanes 3 and 6). After RNase T1 digestion (lanes 2, 3, 5
and 6) samples were resolved by 5% native PAGE. The gel was dried
and exposed to Kodak AR X-ray film with screens at )80 °C for one
day. Swiss 3T3 proteins bound the MARCKS 52 nt cis-element when
transcribed in sense orientation. (B) RNase/EMSA analysis of the
complete MARCKS 3¢-UTR was performed in the presence of the
indicated amounts of the CU-rich element transcribed in sense and
antisense orientation. The nonlabeled sense and antisense pBS-
MARCKS 52 nt transcripts were incubated with 1 lg cytoplasmic
extract from Swiss 3T3 cells for 10 min at room temperature (sense:
lanes 4–9, antisense: lanes 10–14). Then, 2.5 ng of the complete,
32
P-labeled MARCKS 3¢-UTR RNA was added and incubation
prolonged for 20 min at room temperature. Controls were: reaction
buffer (lane 1), BSA (lane 2) and Swiss 3T3 extract without competitor
RNA (lane 3). Undigested probe (lane 1) and RNase T1 digested
samples (lanes 2–14) were loaded on native 5% polyacrylamide gel
followed by electrophoresis at 4 °C. The dried gel was exposed to

X-ray film (Kodak AR) with screens at )80 °C for 1 day. Effective
competition with the MARCKS 3¢-UTR for binding Swiss 3T3 pro-
teins was only detectable by the sense transcript. Note, that the
amounts of the 52 nt antisense RNA was 10-fold higher (lanes 11–14)
than of the respective competitor sense transcript (lanes 5–9). The
positions of the two RNA:protein complexes (C1, C2) are indicated.
(C) Four RNA-binding proteins recognize the MARCKS 52 nt RNA.
In UV-crosslinking experiments with 5 lg extract from Swiss 3T3
cells, proteins of about 30, 36, 40 and 55 kDa were crosslinked to
[a-
32
P]UTP labeled MARCKS CU-rich sense RNA. Specificity of this
interaction was demonstrated by preincubation of the Swiss 3T3
cytoplasmic extract in RNase/EMSA buffer for 10 min with the
indicated amounts of the respective sense DNA oligonucleotide
(employed for cloning of pBS-MARCKS-52nt, see Materials and
methods) prior addition of 2.5 ng
32
P-labeled transcript. After another
10 min incubation at room temperature samples were subjected to
UV-crosslinking (Stratalinker) and RNase A digestion. The denatured
samples (10 min at 95 °C in SDS-sample buffer) were loaded on 12.5%
SDS-polyacrylamide gel and visualized by exposure to X-ray film. The
corresponding antisense probe did not bind any proteins (lane 5).
Position and size of protein markers are indicated on the right.
358 G. Wein et al. (Eur. J. Biochem. 270) Ó FEBS 2003
correlated precisely with the levels of HuR in these samples
(Fig. 5A) supporting the finding that the p36 protein
identified (Figs 3C and 4E) is HuR.
RNase/EMSA analyses demonstrated that the 52 nt

CU-rich RNA can form the complexes C1 and C2 with
both the nuclear (Fig. 5C, lane 1) and the cytoplasmic
fraction (lane 4).
To directly explore the involvement of HuR in forming a
complex with the CU-rich sequence, we used the
Hu-antiserum in combination with RNase/EMSA for
supershift analysis. The nuclear and cytoplasmic extracts
of quiescent Swiss 3T3 cells were preincubated with the
CstF64 antibody 3A7 or with the Hu-specific antiserum.
Following incubation with the labeled MARCKS 52 nt
probe and RNase T1 digestion, the resulting complexes
were resolved by electrophoresis on a native polyacrylamide
gel. The Hu-specific antiserum caused a supershift of the
RNA:protein complexes with the nuclear fraction (Fig. 5C,
lane 2) which was also detectable with cytoplasmic extract,
although much weaker (Fig. 5C, lane 5).
Thus, the Hu-supershift pattern (Fig. 5C) matches accu-
rately the Hu-Western Blot data (Fig. 5A, left panel) and
the UV-crosslinking results (Fig. 5B) further supporting the
finding that HuR is one protein binding the MARCKS
3¢-UTR in Swiss 3T3 cells.
In contrast to the Hu antiserum, no interaction of the
CstF64 control antibody with the MARCKS RNA-binding
proteins was observed revealing that CstF64 is not involved
in formation of the RNA:protein complexes C1 and C2
(Fig. 5B, lanes 3 and 6).
The addition of increasing amounts of the Hu-specific
serum to nuclear Swiss 3T3 proteins resulted in detection of
up to three supershift complexes in addition to the
RNA:protein complexes C1 and C2 (Fig. 5D). It is

therefore likely, that more than one Hu-protein binds the
52 nt CU-rich RNA. Alternatively, the multiple bands may
be due to various post-translational modifications e.g. HuR
protein phosphorylation [56,57].
In summary, the MARCKS 52 nt CU-rich element
represents an effective target sequence for ELAV/Hu-
proteins. One of the proteins of Swiss 3T3 cells binding
the MARCKS CU-rich element was identified as the ELAV
related protein HuR, which is predominately nuclear
localized. CstF64, a further nuclear RNA-binding protein
with sequence specificity for U-residues is not involved in
binding the MARCKS 52 nt CU-rich RNA element in
Swiss 3T3 cells.
Fig. 4. HuD and HuR bind the MARCKS 52 nt CU-rich RNA. (A) For
gel retardation assay, 2.5 ng ( 3000 c.p.m.) of
32
P-labeled MARCKS
52 nt sense RNA was incubated without protein or with the indicated
concentrations of HuD. After 10 min at 37 °C the reaction mix was
resolved on a 0.8% (w/v) agarose gel. The dried gel was exposed to
X-ray film (Kodak AR) at )80 °C for 3 days. Complex formation was
detectable with 0.8 n
M
of GST-HuD protein. (B) Gel retardation assay
was performed with the 52 nt sense CU-rich RNA and GST-HuR; a
RNA:protein complex was formed with 4 n
M
of HuR protein. C: As
negative control, GST-HuD gel retardation assays were done with
labeled pBluescript RNA (pBSD52ntCU-RNA) containing the identi-

cal vector sequences as the 52 nt CU-RNA probe (A, B, D, E) but
without the 52 nt CU-element. No complex formation was detectable
with this probe. D: There was also no complex identified when the 52 nt
CU-rich RNA was incubated with GST. E: UV-crosslinking of
Swiss 3T3 proteins with the CU-rich RNA was performed in the
presence of increasing amounts of recombinant His
6
-HuD. 20 lgof
cytoplasmic extract of Swiss 3T3 cells was incubated with the radio-
labeled MARCKS 52 nt CU-rich transcript (lane 1). Increasing
amounts of recombinant His
6
-HuD was added to the mix, as indicated.
After UV-crosslinking (180 mJ) the samples were digested with
Rnase A and separated by denaturing electrophoresis on a 12.5% SDS-
polyacrylamide gel. Crosslinked proteins were visualized by autoradio-
graphy using Kodak AR X-ray films. Twenty-five nanograms
recombinant His
6
-HuD efficiently blocked the interaction of Swiss 3T3
protein HuR and the RNA probe (crosslinked proteins indicated by
arrows). The positions of protein markers are indicated on the right.
Ó FEBS 2003 Hu-proteins control MARCKS mRNA stability (Eur. J. Biochem. 270) 359
PKC activation causes down-regulation of MARCKS
mRNA in fibroblast but not in neural cells
We recently observed that the MARCKS protein became
phosphorylated in neural PCC7-Mz1 cells upon activation
of PKC [58]; however, the protein was not down-regulated
as demonstrated for murine fibroblasts [22]. Northern Blot
analyses showed that both cell lines expressed the 2.6 kb

mature MARCKS mRNA and more weakly the 4.3 kb
precursor RNA containing an intron of 1.7 kb. RNA
isolated from PCC7-Mz1 cultures treated with solvent or
with PDB (200 n
M
) for 5 h verified that the 2.6 kb
MARCKS mRNA was not down-regulated in PCC7-Mz1
cells by PDB treatment as was the case in Swiss 3T3 cells
analyzed in parallel (Fig. 6A, upper panel). This difference
in regulation of MARCKS mRNA expression was not
Fig. 5. HuR is expressed in the nucleus and binds the 52 nt CU-rich RNA. (A) Quiescent Swiss 3T3 cells were harvested and extracts separated into
nuclear and cytoplasmic fraction by centrifugation (see Materials and methods). Twenty micrograms of each fraction were loaded per lane on a
10% SDS-polyacrylamide gel, separated and transferred to PVDF membrane. Hu-proteins was detected with the affinity-purified Hu-specific
polyclonal antiserum (left panel). For CstF64 detection we applied the 3A7 monoclonal antibody (right panel). Bound antibody was detected by
enhanced chemiluminescence. HuR was predominantly localized in the nucleus, while CstF64 was exclusively localized in the nucleus. (B) For
UV-crosslinking experiments, 20 lg protein of the same extracts as used in Fig. 5A were incubated with the
32
P-labelled 52 nt CU-rich RNA probe
and crosslinked by 180 mJ UV light (Stratalinker). The RNase A digested samples were run on a denaturating, 12.5% SDS-polyacrylamide gel and
the dried gel was exposed to X-ray film (Kodak AR) with screens at )80 °C for 5 days. The band corresponding to the HuR protein is marked by an
arrow. The positions of protein markers in A and B are indicated on the right. (C) Contribution of HuR in formation of the RNA:protein
complexes was revealed by supershift analysis. Ten micrograms of nuclear and cytoplasmic extracts of Swiss 3T3 cells were preincubated with either
the Hu-specific antiserum (4 lL) (lanes 2 and 5), the CstF64-specific antibodies (4 lL) (lanes 3 and 6) or without antiserum (lanes 1 and 4) for
30 min on ice prior addition of 2.5 ng
32
P-labeledMARCKS52 ntCU-richtranscript.TheRNaseT1digestedsampleswereloadedona4%native
polyacrylamide gel and separated at 4 °C. The dried gel was exposed to X-ray film (Kodak AR) at )80 °C for one day. Only the Hu-specific
antiserum caused a supershift of the RNA:protein complexes. (D) Supershift analysis with nuclear Swiss 3T3 extract was performed in the presence
of increasing amounts of the Hu-specific antiserum. Ten micrograms of the nuclear Swiss 3T3 extract (Fig. 5A–C) were preincubated with 1, 2.5, 5
and 10 lL of the Hu-specific antiserum (lanes 2–5) on ice for 30 min prior addition of 2.5 ng

32
P-labeled MARCKS 52 nt CU-rich transcript. The
formation of supershifts were analyzed as described in Fig. 5C. Up to three shifted complexes were observed in addition to complexes C1 and C2.
360 G. Wein et al. (Eur. J. Biochem. 270) Ó FEBS 2003
based on differences in the MARCKS mRNA sequences
because cloning of the MARCKS cDNAs from both cell
lines revealed complete identity (data not shown). Thus,
diverse trans factors expressed in both cell lines seemed to be
responsible for the different regulation of MARCKS
mRNA stability (Fig. 6A).
Having shown that ELAV/Hu-proteins bind the
CU-rich sequence of the MARCKS 3¢-UTR, we investi-
gated whether there is a difference in expression of these
proteins in neural PCC7-Mz1 cells and Swiss 3T3 fibro-
blasts. Only the HuR protein of 36 kDa was detected in
Swiss 3T3 extracts (20 lg) by Western blot analysis using
the Hu-specific antiserum (Fig. 6B). In PCC7-Mz1 cells
(10 lg extract was loaded) levels of HuR were about
fivefold higher than in Swiss 3T3 cells. Furthermore, in
extracts of neural PCC7-Mz1 cells additional bands of
approximately 38–42 kDa were detectable, corresponding
to neuronal members of ELAV/Hu-family (Hel-N1, HuC
and HuD) [59]. Staining of all bands could efficiently be
prevented by preincubation of the Hu-antiserum with
recombinant His
6
-HuD fusion protein showing the speci-
ficity of the detected bands (data not shown). Performing
RNase/EMSA experiments with the CU-rich sequence and
PCC7-Mz1 extracts (data not shown) revealed a third

complex in addition to the two described complexes C1
and C2 (Figs 2B and 3A). Furthermore, in UV-crosslink-
ing analysis with PCC7-Mz1 extracts (data not shown) a
complex of about 39 kDa, not detected with Swiss 3T3
extracts (Fig. 4E, lane 1), became evident, which was of the
same size as the complex between His
6
-HuD and the 52 nt
CU-rich RNA (Fig. 4E, lanes 2–7).
Based on these results, it was tempting to speculate that
the high expression of members of the ELAV/Hu family is
responsible for the MARCKS mRNA stability in PCC7-
Mz1 cells.
Role of ELAV/Hu in controlling stability
of MARCKS mRNA
We have demonstrated that ELAV/Hu proteins have a high
affinity to the identified 52 nt CU-rich cis-element within
the 3¢-UTR of the MARCKS mRNA. Furthermore, we
could show that PCC7-Mz1 cells express neuronal specific
members of the ELAV/Hu family in addition to high levels
of HuR (Fig. 6B). To elucidate whether the neuronal HuD
has a direct effect on controlling MARCKS mRNA
stability, we cloned the cDNA coding for HuD into the
eukaryotic expression vector pTRE. The promoter activity
is controlled by the tetracycline-controlled transactivator
(tTA). This construct (pTRE-HuD) was transiently trans-
fected into Swiss 3T3 cells together with the pTetoff plasmid
coding for the tTA-protein. Under these conditions the
PDB initiated down-regulation of the MARCKS mRNA
was completely blocked (Fig. 7, lane 4). A similar stabiliza-

tion of the MARCKS mRNA after PDB treatment was also
observed when HuR was transiently overexpressed in
Swiss 3T3 cells (lane 6). The level of the MARCKS mRNA
was even elevated in nontreated HuD and HuR-transfected
cells (Fig. 7, lanes 3 and 5).
These data clearly demonstrate that the MARCKS
mRNA can be stabilized by members of the ELAV/Hu
gene family. Its high expression in PCC7-Mz1 cells seems to
Fig. 6. Down-regulation of MARCKS mRNA in Swiss 3T3 fibroblasts
but not in neural PCC7-Mz1 cells upon phorbol ester treatment. (A)
PCC7-Mz1 cells were seeded at a density of 1.75 · 10
4
cm
)2
and the
following day treated with PDB (200 n
M
,5 h).Swiss 3T3cells(1 · 10
5
cells) were seeded on 90-mm cell culture dishes. After 1 week, when
cultures reached confluence, cells were treated with 200 n
M
PDB for
5 h. For Northern blotting, total RNA was isolated and 5 lgRNA
separated per lane on a 1.2% (w/v) agarose/2.2% (v/v) formaldehyde
gel. After transfer on a nylon membrane by capillary blot the RNA was
hybridized with a
32
P-radiolabelled MARCKS cDNA probe (p809.1)
[22], washed and exposed to X-ray film (Kodak AR) with intensifier

screens at )80 °C for 2 days (upper panel). PDB treatment caused
down-regulation of MARCKS mRNA in murine fibroblasts
(Swiss 3T3) but not in neural precursor cells (PCC7-Mz1). Ethidium
bromide staining shows equal loading of the gel and integrity of the
RNA (lower panel). The positions of 18S and 28S rRNA are indicated.
(B) Western blot analysis with the affinity-purified Hu-specific poly-
clonal antiserum was performed. The following samples were loaded
and separated in a 10% (w/v) SDS-polyacrylamide gel: 10 lgprotein
of untreated (–PDB) and PDB-treated (+PDB) (200 n
M
PDB, 5 h)
PCC7-Mz1 stem cells, 20 lg protein from extracts of untreated (–PDB)
or PDB-treated (200 n
M
PDB,5h)(+PDB)Swiss3T3cells,asindi-
cated. After gel electrophoresis, proteins were transferred onto PVDF-
membrane. Hu-proteins were detected with the Hu-specific antiserum
and visualized by enhanced chemiluminescence. Because of the high
degree of sequence identity between members of the ELAV/Hu-family
(about 70%) [78], the Hu-antiserum recognizes several members of this
family. In Swiss 3T3 cells, only one band of 36 kDa was recognized,
corresponding to HuR. In neural PCC7-Mz1 cells, additional
Hu-proteins were detected demonstrating the expression of several
members (e.g. HuC, HuD and Hel-N1) of the ELAV/Hu gene family.
Ó FEBS 2003 Hu-proteins control MARCKS mRNA stability (Eur. J. Biochem. 270) 361
be responsible for constitutive stabilization of the
MARCKS mRNA.
Discussion
The regulation of mRNA decay is a major control point in
gene expression. In the present study we show that: (a) a

region containing an element of 52 nucleotides
[(CUUU)
11
U
8
]ofthe3¢-UTR of the MARCKS mRNA
confers RNA instability when merged with a reporter gene;
(b) this CU-rich sequence of 52 nucleotides of the
MARCKS 3¢-UTR is specifically bound by members of
the ELAV family (HuD, HuR); (c) upon activation of PKC,
stability of the MARCKS mRNA is drastically reduced in
murine fibroblasts, but not in neural stem cells expressing
high levels of Hu-proteins; and (d) overexpression of HuD
and HuR in fibroblasts caused a drastic stabilization of the
MARCKS mRNA even when PKC was activated.
The identified CU-rich sequence of the 3¢-UTR of the
MARCKS mRNA belongs to the AU-rich elements (ARE)
previously found in the 3¢-UTRs of some short-living
messengers, such as those for cytokines and lymphokines.
AREs can be divided into three classes based on their
structural and functional properties. Class I and II AREs are
characterized by the presence of the pentanucleotide
AUUUA, which is absent in class III AREs. One to three
copies of the pentanucleotide AUUUA are either distributed
over the entire 3¢-UTR,e.g.c-fos (class I) or three or more
AREs are located in tandem, e.g. TNFa (class II) [9,10]. The
mRNAs with class III AREs (e.g. c-jun) do not contain the
AUUUA pentanucleotide but only U-rich segments [60].
Because the CU-rich element of the MARCKS 3¢-UTR
causes RNA instability but does not contain the character-

istic AUUUA element, we identified herewith a novel ARE
motif belonging to the AREs of class III.
So far, 15 different proteins have been shown by
UV-crosslinking and gel-shift assays to recognize AU- and
U-rich RNA sequences: AUBF, AU-A (HuR) [61], AU-B,
AU-C, Hel-N1, hnRNP D (AUF1), hnRNP A1, hnRNP C,
AUH, GAPDH, hnRNP A0, HuD, tristetraprolin and
TIAR. However, only three of these proteins, hnRNP D
and HuR and tristetraprolin seem to influence stability of
ARE-containing mRNAs in vivo (reviewed in [51,62]). It is
likely that HuR is involved in controlling the stability of the
MARCKS mRNA.
Our data show that HuR binds with high affinity to the
MARCKS ARE. Furthermore, both elevated levels of endo-
genous Hu proteins as in neural PCC7-Mz1 cells and overex-
pression of HuD/HuR in fibroblasts dramatically stabilize
the MARCKS mRNA. Thus, the MARCKS mRNA is an
additional example that Hu-protein overexpression stabilizes
ARE-containing mRNAs [16,17,52,53,63]. Presently we can
only speculate on the mechanism by which ELAV/Hu
proteins control the MARCKS mRNA stability. A working
model hypothesizes that the CU-rich element confers insta-
bility by recruiting RNases to the MARCKS mRNA, and
that binding of Hu-proteins prevents this effect. Therefore,
one might envisage that the affinity of HuR to the MARCKS
mRNA in Swiss 3T3 cells is controlled by protein kinases, as
was recently found for several AREs [56,57].
Our preliminary data show no difference in the capability
of the 52 nt CU-rich element to form the complexes C1 and
C2 with extracts of untreated and PDB-treated fibroblasts.

This might be due to the fact that in vitro binding of the
52 nt CU-element by ELAV proteins may not reflect the
physiological situation, i.e. treatment with PDB. This
interaction might be highly regulated intracellularly for this
ARE in a cell type- and physiological state-dependent
manner as has recently been shown for several AREs [80].
Thus, four mammalian ligands, three of them known to be
protein phosphatase 2A inhibitors, were recently identified,
which modulate HuR’s ability to bind its target mRNAs
in vivo [64]. Other cis elements of the MARCKS mRNA,
outside of the 3¢-UTR, and their corresponding trans factors
may additionally contribute to control binding of ELAV
proteins and consequently mRNA stability.
Recent studies show that HuR binds ARE-containing
mRNAs in the nucleus and transports them as RNPs to the
cytosol [16,22,65,75]. It is likely that HuR together with
other factors is involved in nucleo-cytoplasmic shuttling of
the MARCKS mRNA. An alternative explanation for the
described stabilization of the MARCKS mRNA observed
in 3T3 cells overexpressing HuR and HuD (Fig. 7) is that
these proteins are active in decay, but when overexpressed
they sequester other factors needed for RNA degradation.
Antisense RNA- or RNAi-experiments to ablate HuR
expression will help to elucidate this question.
Perturbations in the 3¢-UTR-mediated regulation was
shown to cause loss of control over one or more genes.
Several disorders, such as carcinoma, inflammation, myto-
tonic dystrophy, a-thalassemia (reviewed in [66]) and
Morbus Alzheimer [3,67] are caused by mutations in the
Fig. 7. Transient overexpression of HuD and HuR stabilizes the

MARCKS mRNA. Swiss 3T3 cells were plated (3.5 · 10
5
cells per
90-mm dish) and transfected the following day with the plasmid
pTetoff together with the empty pTRE vector (lanes 1, 2) or together
with pTRE-HuD (lanes 3, 4) or together with pTRE-HuR (lanes 5, 6)
by lipofection. After one day cultures remained untreated (–PDB) or
were treated with 200 n
M
PDB for 5 h (+PDB). Total RNA was
isolated and 5 lg loaded on a 1.2% (w/v) formaldehyde/agarose gel for
NorthernblotanalysisasdescribedinlegendtoFig.6A.Thedegra-
dation of the MARCKS mRNA by activation of PKC by PDB
treatment (lane 2) was completely blocked by overexpression of HuD
(lane 4) and HuR (lane 6) (upper panel). Staining of the gel with
ethidium bromide revealed equal loading of the gel and integrity of the
RNA (lower panel). The positions of the 28S and 18S rRNAs are
indicated on the left.
362 G. Wein et al. (Eur. J. Biochem. 270) Ó FEBS 2003
3¢-UTR sequences or in the 3¢-UTR-binding regulatory
proteins. Neoplastic transformation has been shown to
stabilize ARE-containing mRNAs [68] and has been
associated with the activation of c-Jun N-terminal kinase
(JNK) [69]. Similarly, activation of MAP kinase-activated
protein kinase 2 has been associated with the stabilization of
ARE-containing mRNA in HeLa cells [70].
Because MARCKS emerges as a growth and tumor
suppressor gene [23,25], which is down-regulated in many
transformed cell types, it will be of interest to investigate the
reasons for its low expression in malignant cells. It is

possible that mutations in its 3¢-UTR prevent binding of
factors like HuD/HuR which normally inhibit degradation
of the transcript. Alternatively, the MARCKS gene might
be transcriptionally down-regulated as recently shown for
immortalized rat hippocampal cells [71].
Elucidating the exact mechanism by which ELAV proteins
confer stability to the MARCKS mRNA will help to identify
other genes following the same mechanism of regulation. In
search for such potential tumor suppressor genes, the
sequence of the identified 52 nt cis-element will be useful.
However, RNA-binding proteins recognize both the primary
sequence and the secondary structure [48,72–74]. In this
context it is interesting to mention that the computer
programs
MFOLD
and
MPLOT
(German Cancer Research
Institute Heidelberg, Germany) [76,77] predicted an
extremely stable secondary structure for the MARCKS
3¢-UTR (DE ¼ )412.2 kJ at 22 °C) and that the 52 nt
CU-sequence forms two helical and one stem-loop structure
(data not shown). More than 70% of all bases of the 52 nt
CU-rich element form double helical structures. This robust
conformation is maintained at 22 °Caswellas37°Cand
may be necessary for the function of the CU-element, e.g. for
protein binding. Therefore, the secondary structure of the
MARCKS 3¢-UTR has to be taken into account when
searching for genes regulated in the same fashion. It is now
becoming apparent that the combination of functionally and

structurally distinct sequence motifs, such as AU-pentamers,
nonamers and U-rich stretches, determines the ultimate
destabilizing ability of each individual ARE. Knowing the
higher structural order, the entire cis elements, and all trans
factors, including their regulation and post-translational
modifications like phosphorylation, of the MARCKS
mRNA will be necessary to understand the linkage of cell
signaling pathways to the mRNA degradation machinery.
This knowledge may aid in the design of novel approaches to
the therapeutic intervention in the various abnormalities that
are associated with deregulated mRNA stability.
Acknowledgements
We wish to thank Dr I. Mattaj (EMBO, Heidelberg) for providing
CstF64 antibody, Dr H. Kleinert and Dr F. Scha
¨
fer for making DNA
constructs available, and Dr A. Maelicke (all University of Mainz) for
his encouragement and support. Financial help by a grant of the DFG
(He 2557/2-1) is gratefully acknowledged. This work represents parts of
the PhD theses of G. W. and R. K.
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