Genomic organization of
MUC4
mucin gene
Towards the characterization of splice variants
Fabienne Escande
1,2,3
, Laurent Lemaitre
1
, Nicolas Moniaux
4
, Surinder K. Batra
4
, Jean-Pierre Aubert
1,2
and Marie-Pierre Buisine
1,2,3
1
INSERM Unite
´
560, Lille, France;
2
Laboratoire de Biochimie et Biologie Mole
´
culaire, Ho
ˆ
pital C. Huriez,
Centre Hospitalier Re
´
gional et Universitaire, Lille, France;
3
Faculte

cells. They contain a high percentage of threonine and serine
residues carrying O-linked glycan chains and distributed in
tandemly repeated motifs in the central part of the protein
backbone. Mucins are known to play important roles in the
lubrication and protection of mucosae but more recently,
the involvement of mucins in the renewal and differentiation
of the epithelia, cell adhesion and cell signaling has also been
proposed [1–3].
To date, 14 human mucin genes have been identified:
MUC1–4, MUC5B, MUC5AC, MUC6–8, MUC11–13,
MUC16 and MUC17 [4–6]. MUC4 is a member of the
membrane-bound mucin family and is believed to be the
homologue of the rat sialomucin complex (SMC, rat Muc4)
because of their similar structural organization [7–10]. Rat
Muc4 is a well-characterized heterodimeric glycoprotein
complex in which the mucin subunit ascite sialo-glycoprotein
(ASGP)-1 is the major detectable glycoprotein. The other
subunit ASGP-2 is membrane-associated and contains
epidermal growth factor (EGF)-like domains that were
shown to act as a ligand for the tyrosine kinase p185
neu
[11].
The full cDNA of MUC4, also called sv0-MUC4,was
entirely characterized in our laboratory [10,12,13]. The
deduced amino-acid sequence of the N-terminal region
contains a peptide signal, followed by three imperfect
repetitions of a motif, varying from 126 to 130 residues,
and by a unique threonine- and serine-rich sequence. The
central region is composed of a large mucin-type domain
characterized by the perfect repetition of 16 amino-acid

Note: the nucleotide sequences reported here have been submitted to
EMBL Nucleotide Sequence Database under accession numbers
AJ430032, AJ430033, and AJ430034.
(Received 8 February 2002, revised 30 May 2002,
accepted 31 May 2002)
Eur. J. Biochem. 269, 3637–3644 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03032.x
deletions and/or insertions located in the 3¢ region but also
for two of them by deletion of the central repetitive domain.
Until now, because of the lack of knowledge on the genomic
organization of the 3¢ region of MUC4, the precise mech-
anisms responsible for these events could not be defined.
In the present paper, we described the genomic structure
of the 3¢ region of the human MUC4 gene. A comparison of
the nucleotide genomic and cDNA sequences allowed us to
establish the nature of the mechanisms responsible for the
diversity of the MUC4 transcripts.
EXPERIMENTAL PROCEDURES
Oligonucleotide primers
Oligonucleotides used for PCR are shown in Table 1. They
were synthetized by Eurogentec (Lie
`
ge, Belgium) or by
MWG-Biotech (Ebersberg, Germany).
PCR amplification of human MUC4 introns
MUC4 introns were amplified from a bacterial artificial
chromosome (BAC) clone containing the human MUC4
gene [14]. Amplifications were performed in a PerkinElmer
Thermal Cycler 2400 (Applied Biosystems, Courtaboeuf,
France). PCR reactions were conducted in 50-lL reaction
volumes, containing 1 lg of BAC DNA, 5 lLof10·

,10pmolofeachprimer
and 2.5 U of DNA polymerase. The cycle parameters were
94 °C for 2 min, followed by 30 cycles at 94 °Cfor10s,
annealing at 60 °C for 45 s, and elongation at 71 °Cfor
2min,and68°C for 10 min. The last 20 cycles had their
elongation time extended by 10 s for each new cycle. The
final elongation step was extended for an additional 15 min
Table 1. Primers used for DNA amplification and sequencing.
a
Nucleotide position is defined according to the sequence of sv0-MUC4 (AJ010901).
b
S, sense; AS, antisense.
3638 F. Escande et al. (Eur. J. Biochem. 269) Ó FEBS 2002
at 71 °C. PCR products were analyzed by 1% agarose gel
electrophoresis and cloned directly into the pCR2.1 vector
with the original TA cloning
TM
kit (Invitrogen, Leek, the
Netherlands), according to the manufacturer’s instructions.
Plasmid DNA purification
Plasmid DNA was purified using the QIAprep Spin Plasmid
kit (Qiagen, Courtaboeuf, France).
DNA sequence analysis
Sequences were determined by automatic sequencing with a
DNA sequencer model 4000 L LI-COR and the Sequi-
Therm Excel
TM
II Long-read Premix DNA sequencing Kit-
LC (TEBU, Le Perray en Ivelynes, France), using standard
vector primers or with ABI PRISM model 377 XL

which was exclusively reported for the sv1-variant (Fig. 1).
Exon and intron sizes, splice junction types and sequences of
the exon–intron boundaries are given in Table 2. The size of
the 24 introns ranged from 94 to 2.8 kb, and the size of the
24 exons ranged from 65 to 607 bp. All of the 5¢ donor and
3¢ acceptor sites were consistent with the consensus gt–ag
motifs described for splice sites in eukaryotic genes [24].
Unique tandemly repeated sequences, more and less perfect,
were found in some introns: approximately 90 copies of an
15-bp repeat (AGGTATGGGTGTGGA) in intron 3,
approximately 60 copies of an 26–31 bp repeat in intron 4,
23 copies of a 32-bp repeat (CAGGAGTACCCCA), four4
copies of a 34-pb repeat (AGGCCTCAACACCCCCC
AGCACCTTCCCCAGGCC) in intron 23. A search of the
GenBank database indicated that the consensus sequences
of these four repeat were not identical with any other
genomic sequence. Sequence type microsatellites were also
found in others introns: (GGT)
124
in intron 7 (T/CG)
22
in intron 16 (GATA)
73
in intron 18. Such repetitive
intronic sequences may participate with the repetitive
sequence in the central exon to interindividual polymorph-
ism and may be used as a potential intragene marker of the
locus 3q29.
Therefore, with the previously reported first two exons,
the MUC4 genomic organization is complete: MUC4 is

consensus splice donor/acceptor sites [25] and demonstrated
a remarkable similarity with the splice donor/acceptor
sequences observed in sv0-MUC4.
A small part of the events recently described by Chou-
dhury et al. [22] could not be explained. These events should
result from more complex mechanisms.
DISCUSSION
The human MUC4 belongs to the mucin family. Like the
other members of this family, MUC4 is found in the mucus
Fig. 1. Organization of the 3¢ region of human MUC4 gene. Boxes
indicate exons. They are numbered consecutively above the boxes
with 2 for the central exon (black box). Shaded grey box indicate
3¢-untranslated region. Horizontal lines indicate introns. They are
numbered below the lines. The length of the exons and introns are
showntoscale.
Ó FEBS 2002 Splice variants of MUC4 (Eur. J. Biochem. 269) 3639
secretion and corresponds to a high molecular mass
O-glycoprotein. It exhibits a VNTR polymorphism corre-
lated with the variation of one unit of repetition that
composes its central domain. In opposition with the strictly
secreted mucins, several transcripts of MUC4 were previ-
ously identified. This property, to be expressed under
numerous RNA forms for human mucins, appears to be
shared by the members of the membrane-bound mucin
subfamily. Indeed, four distinct transcripts were character-
ized for MUC1 as well as for MUC3 [26–30]. In both cases,
the perfect knowledge of the genomic organization allowed
the authors to assimilate transcripts with alternative splice
forms of the MUC1 and MUC3 genes.
Right now, MUC1 is the best known and characterized

important role in cell proliferation and differentiation of
epithelial cells. A secreted form was identified for rMuc4;
however, it was shown not to be caused by alternative
splicing of its premRNA but by proteolytic cleavage of the
membrane-bound mucin [36]. Therefore at this point, only
one transcript was isolated for rMuc4,aswellasfor
mMuc1, mMuc3,andrMuc3.
In this study, we determined, for the first time, the
genomic organization of the MUC4 gene as well as the
mechanisms responsible for the diversity of MUC4 tran-
scripts. We have demonstrated that the events (deletions/
insertions) observed in the 24 MUC4 variants are generated
by different mechanisms of alternative splicing: alternative
use of exons, which is the mechanism most commonly used
to generate isoforms, and the use of cryptic donor/acceptor
splice sites. The identification of the same event in several
MUC4 variants, isolated from different tissues or cell lines,
and the molecular characterization of the splice events
strongly suggest that the diversity in the 3¢ region of MUC4
variants is not due to an error in the splicing process or an
artifact.
Altogether, it appears that the membrane-bound mucins
possess, at least from the transcriptional point of view, a
level of complexity in more than their animal homologues.
This complexity seems to be the highest for MUC4,asingle
gene code for at least 24 distinct transcripts. As no evidence
Table 2. Characteristics of the exon–intron junctions of the MUC4 gene.
3640 F. Escande et al. (Eur. J. Biochem. 269) Ó FEBS 2002
has been available to confirm the translation of the
transcripts, it is difficult to study their function. If translated,

domain; CT1-CT12, C-terminal domains as described in [10].
3642 F. Escande et al. (Eur. J. Biochem. 269) Ó FEBS 2002
ACKNOWLEDGEMENTS
This work was supported by the Association de Recherche contre le
Cancer and a RO1 grant from the National Institutes of Health
(CA78590). We gratefully acknowledge D. Demeyer, C. Mouton,
M. Cre
´
pin for performing automatic sequences and A. Bernigaud,
D. Petitprez and V. Mortelec for the excellent technical assistance.
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