Báo cáo khoa học: Complex alternative splicing of the hKLK3 gene coding for the tumor marker PSA (prostate-specific-antigen) ppt

Complex alternative splicing of the hKLK3 gene coding
for the tumor marker PSA (prostate-specific-antigen)
Nathalie Heuze
´
-Vourc’h, Vale
´
rie Leblond and Yves Courty
Laboratoire d’Enzymologie et Chimie des Prote
´
ines, EMI-U 0010, Universite
´
F. Rabelais, Tours, France
PSA (prostate-specific antigen), the most useful serum
marker for prostate cancer, is encoded by the hKLK3 gene
and is present in the serum as a mixture of several molecular
species. This work was performed to identify the hKLK3
transcripts in order to determine how many proteins
resembling PSA are synthesized from the hKLK3 gene and
secreted in blood. Combined Northern blotting, molecular
cloning and database searching showed that the hKLK3 gene
produces at least 15 transcripts ranging in size from 0.7 to
6.1 kb. Polysomal distribution analysis revealed that the
transcripts shorter than 3.1 kb are efficiently translated in
prostate cell line. A total of 12 hKLK3 transcripts have been
completely or partially cloned. They result from alternative
splicing or/and alternative polyadenylation involving com-
plex regulation. They code for eight proteins: PSA, a trun-
cated form of PSA (PSA-Tr), five PSA variants (PSA-RPs)
and one protein (PSA-LM) unrelated to PSA. Using a spe-
cific antibody, we detected the PSA-RP2 variant in prostate
tissue. All the variants share the same signal peptide and

could contribute to the diversity of hKLK3 proteins in
prostate fluid and blood.
Keywords: alternative mRNA; PSA variant; tumor marker;
prostate cancer.
PSA (prostate-specific antigen) is encoded by the hKLK3
gene, which belongs to the tissue kallikrein gene family
located at chromosome locus 19q13.3–19q13.4 [1,2]. PSA
(also named hK3) is a serine protease abundantly produced
by human prostate epithelial cells. This protein is secreted
into the lumina of prostate ducts and is present at very high
concentrations in the seminal plasma (reviewed in [3]). PSA
hydrolyses semenogelins I and II, resulting in liquefaction of
the seminal plasma clot after ejaculation [4]. Although it
seems modulating the proliferation of normal and malig-
nant cells and the angiogenesis [5–8], the role of PSA in
prostate pathologies remains unclear.
PSA is presently considered to be the best available
marker of prostate tumors, and is widely used for screening,
diagnosing and monitoring prostate cancer (PCa) [9,10].
Nevertheless, concentrations of PSA below 10 ngÆmL
)1
do
not distinguish between Pca and benign prostatic hyper-
plasia (BPH). Various molecular forms of PSA are present
in the serum, some of them being complexed with serine-
protease inhibitors while the others are uncomplexed or free
[11]. It is important to identify each of the free forms, as the
proportions of some of them differ in BPH and in cancer
[12]. It has been recently demonstrated that some of the free
forms are produced by proteolysis of proPSA [13] or mature

PSA [14]. Some of the others could be produced by
alternative splicing [15].
Alternative splicing is the most widely mechanism used to
enhance protein diversity, and could affect the product of
over 35% of human genes. Multiple hKLK3 transcripts
were detected by Northern blot analysis [16,17], but most
investigations have focused on PSA produced from the
major 1.6 kb mRNA. This work was performed to identify
the numerous hKLK3 transcripts, and then determine how
many proteins resembling PSA can be synthesized by the
hKLK3 gene. This report describes the complete or partial
characterization of 12 hKLK3 transcripts produced by
multiple splicing or polyadenylation. They code for at least
eight proteins. Some of them are variants of PSA and
appear to be good candidates for identifying free circulating
species.
Materials and methods
Samples and RNA isolation
The LNCaP cell line (American Type Culture Collection,
ATCC CRL-1740) was derived from human metastatic
adenocarcinoma of the prostate. Cells were grown in
RPMI-1640 (Life Technologies SARL, Cergy Pontoise,
France) supplemented with 5% (w/v) fetal bovine serum,
100 UÆmL penicillin/streptomycin, 2 m
M
glutamine in the
presence of the synthetic androgen R1881 (0.1 n
M
;NEN-
Dupont, Les Ulis, France) [15,18]. Tumor specimens were

obtained with informed consent from patients undergoing
transurethral prostatectomy. Total and poly(A) RNA
were prepared as previously described [18]. Normal
prostate total RNA was from BD Clontech (Palo Alto,
CA, USA).
Correspondence to Y. Courty, Laboratoire d’Enzymologie et Chimie
des Prote
´
ines, EMI-U 0010, 2 bis bd Tonnelle
´
, 37032 Tours, France.
Fax: + 33 2 47 36 60 46, Tel.: + 33 2 47 36 60 50,
E-mail:
Abbreviations: BPH, benign prostate hyperplasia; hK or hKLK,
human kallikrein; Pca, prostate cancer; pISE, putative intron splicing
enhancer; PSA, prostate-specific antigen; PSA-LM, PSA-linked
molecule; PSA-RP, PSA-related protein; PSA-Tr, PSA truncated;
CAPS, [cyclohexylamino]-1-propanesulfonic acid.
(Received 18 October 2002, revised 6 December 2002,
accepted 11 December 2002)
Eur. J. Biochem. 270, 706–714 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03425.x
Polysomal RNA preparation
LNCaP cells (5 · 10
8
) were cultured for four days in the
conditions described above and centrifuged at 1000 g for
10 min at room temperature. Cells collected on ice were
diluted in 1 mL of a buffer (50 m
M
Tris/HCl pH 8.0,

250 m
M
KCl, 5 m
M
MgCl
2
) containing 250 m
M
sucrose,
2m
M
dithiothreitol, and 3 mg of yeast total RNA (Roche
Diagnostics, Meylan, France). The cells were dounce homo-
genized with 10 strokes of a type B pestle and centrifuged at
10 000 g for 10 min at 2 °C. The supernatant was supple-
mented with 2% Triton X100 and 0.2 mgÆmL
)1
of heparin
and incubated on ice for 10 min. The supernatant was
layered on top of a 10–50% sucrose gradient and centri-
fuged at 40 000 r.p.m. for 50 min at 2 °CinanL5
ultracentrifuge (Beckman) equipped with an SW41 rotor.
Samples of 500 lL were collected from the sucrose gradient,
and 250 m
M
EDTA and 0.5% SDS were added to each
fraction. RNA was then purified using 500 lL phenol/
chloroform (1 : 1, v/v) and ethanol precipitated. The pellets
were dissolved in DEPC-treated H
2

O and stored at )70 °C.
Spectrophotometric RNA quantification was performed on
an aliquot of each sample.
Probes and hybridization
A 42-base 5¢-biotinylated oligonucleotide corresponding to
a part of exon 2 of the hKLK3 gene (position 1760–1801,
EMBL X14810) was used as template to synthesize an
antisense [a
32
P]-labeled probe using the Klenow fragment of
Escherichia coli DNA polymerase I. After heat denatura-
tion, the biotinylated unlabeled strand was captured using
Streptavidin MagneSphereÒ Paramagnetic Particles
(Promega Corp., Madison, WI, USA). The labeled strand
was recovered for Northern blot hybridization.
Various hKLK3 gene fragments were obtained from the
LNCaP cDNA library by PCR amplification using Pro-HA
DNA polymerase (1.25 U, Eurogentec, Seraing, Belgium).
The PCR reactions involved heating at 94 °Cfor2minand
30 cycles of 94 °C for 30 s, annealing temperature (Table 1)
for 30 s and 75 °C for 1.5 min. The resulting fragments
were purified using the WizardÒ PCR preps DNA purifi-
cation system (Promega Corp.) and 50–100 ng were labeled
with [a
32
P]dCTP by random priming.
Northern blot hybridization was performed overnight at
68 °C with the QuikHyb Hybridization solution (Strata-
gene,LaJolla,CA,USA).Blotswerewashedat68°Cfor
2 · 30minin2· NaCl/Cit, 0.1% SDS and 2 · 20 min in

0.1 · NaCl/Cit, 0.1% SDS. Membranes were then exposed
to Kodak AR X-ray film at )70 °C using intensifying
screens from 4 h to 6 days.
Rapid amplification of cDNA ends and DNA sequencing
The hKLK3 cDNA clones were obtained by 5¢ and/or 3¢
rapid amplification of cDNA ends (RACE) using the
Marathon cDNA Amplification Kit (BD Clontech) Mara-
thon cDNAs were generated from LNCaP poly(A) RNA
[15], and from tissular poly(A) RNA according to the
manufacturer’s instructions. RACE-PCR was carried out
with an hKLK3-specific primer (K3-PCR2: 5¢-CAC
CCGGAGAGCTGTGTCACC-3¢) based on a sequence
just downstream of the transcription initiation site of the
hKLK3 gene and the Marathon adaptor primer 1 (AP1)
using the Expand Long Template PCR System (Roche
Diagnostics). The thermocycling protocol was: denatura-
tion at 94 °C for 2 min; 5 cycles of denaturation at 94 °Cfor
30 s, annealing and elongation at 72 °C for 3.30 min; 5
cycles of denaturation at 94 °C for 30 s, annealing and
elongation at 70 °C for 3.30 min; 25 cycles, 94 °Cfor30s,
Table 1. Primers used for PCR. Localization of the primers (intron/exon) refers to the structure of the major transcript.
Primer pair Localization Primer sequence (5¢fi3¢)
PCR product size
(bp)
Annealing temperature
for PCR (°C)
Probes
K3-540 Intron 1 AACCCAGCACCCCAGCCCAGACAG 610 65
K3-1100 CAGAGAGCTGGCAGTTGTGGCTGG
K3-2016 Intron 2 CATGGCTGCCTGGGTCTCCATCTG 286 65

K3-2300 CAGGCCCATCCCGTGCGTGTG
K3-3500 Exon 3 AAGCGTGATCTTGCTGGGTCGGCA 287 65
K3-3757 CACTCCTCTGGTTCAATGCTGCCC
K3-int3 Intron 3 GAGTGTACGCCTGGGCCAGATG 118 55
K3-0.7rev2 TGGACCCCACCTGGTTCCTTGG
K3-5016 Intron 4 TGCCGATGGTCCTCCAT 346 50
K3-1.7rev CTATCTTTCAGACCTGGACAGGC
RT-PCR
K3-PCR2 with Exon 1 CACCCGGAGAGCTGTGTCACC
K3-1.5 Exon 5 GGGGTTGGCCACGATGGTGTC 798 (PSA) 68
K3-0.7rev2 Intron 3 TGGACCCCACCTGGTTCCTTGG 622 (PSA-RP2) 68
K3-5055 Intron 4 GACACCTCCTCTCCAGGGCAC 731 (PSA-RP1) 68
K3-MU1 Exon3alt CTCCTCTGGTTCAATGCTGGAG 352 (PSA-RP4) 68
SSI (with K3-1.5) Exon2-Exon3 CTGCCCACTGCATCAGGAAGC 463 (PSA-RP3) 68
K3-Ex1 with Exon 1 TTACCACCTGCACCC 532 (PSA-RP5) 51
K3-sp5 Exon4-Intron4 GGTCAAGAACTCCTCTG 53
Ó FEBS 2003 Complex splicing of hKLK3 (Eur. J. Biochem. 270) 707
68 °C for an initial duration of 3.30 min and an automatic
increment of 20 s at each cycle. The cDNA encoding PSA-
RP3 was obtained using a 5¢-anda3¢-RACE performed
with the following primer pairs: AP1 and SSI-rev (5¢-
TGGAGTCATCACCTGGCTTCC-3¢), and AP1 and
SSI (5¢-CTGCCCACTGCATCAGGAAGC-3¢). Amplified
products were cloned into a pCR 3.1 vector and trans-
formed TOP10F¢ competent cells (Invitrogen, Breda, the
Netherlands). DNA was sequenced on both strands with an
automated sequencer (ABI prism DNA 377 sequencer,
Perkin Elmer).
Expression analysis of splice variants
Expression of splice variants was analyzed in prostate

samples by RT-PCR. cDNA was synthesized from 5 lg
total RNA using SuperScript II reverse transcriptase
(Invitrogen) according to the manufacturer’s instructions.
PCR was performed using specific primers (Table 1) with
the following cycling conditions: 94 °Cfor3minand35
cycles at 94 °C for 30 s, 68 °Cor55°C for 30 s and 72 °C
for 75 s. The products were electrophoresed on 1% (w/v)
agarose gels and visualized by ethidium bromide staining.
DNA corresponding to the major PCR product was
extracted from the agarose gel and sequenced.
Production of polyclonal peptide antibodies
and protein analysis
A PSA-RP2 oligopeptide corresponding to amino acids
165–180 of the putative prepro PSA-RP2 was synthesized
and purified by high-performance liquid chromatography.
The peptide was conjugated with BSA and used to
immunize rabbits. The anti PSA-RP2 Ig was purified by a
recombinant PSA-RP2 peptide-affinity column.
Cancer prostate tissue (100 mg) was pulverized in liquid
nitrogen to a fine powder, 1.5 mL of TRIzol reagent
(Life Technologies SARL) added and the proteins extrac-
ted according to the manufacturer’s conditions. Recom-
binant PSA-RP2 was from a cytosolic extract of CHO
cells (Chinese hamster ovary cell line, ATCC CCL61)
stably transformed with an expression vector containing
the entire sequences encoding prepro-PSA-RP2 [18].
Proteins were separated by SDS/PAGE on a 12% gel
under reducing conditions and electrotransferred to a
poly(vinylidene difluoride) membrane (Millipore Corp.,
Bedford, MA, USA) in a [cyclohexylamino]-1-propane-

sulfonic acid (CAPS) buffer (Sigma-Aldrich Corp., St
Louis, MI, USA) [18]. ECL Western analyses were
carried out following the supplier’s instructions (Amersham
Life Sciences, Les Ullis, France) using the anti-RP2
antibody described earlier. The second antibody was
peroxidase-conjugated mouse antirabbit immunoglobulins
(Sigma-Aldrich Corp.).
Results
Expression pattern of the hKLK3 gene in prostate
Prostate tissue contains a major 1.6-kb transcript (K3a) that
encodes hK3/PSA (Fig. 1A). Several other hybridization
bands in the 6.1–0.6 kb range were detected with the exon 2
probe, albeit at much lower levels. Poly(A) RNA gave a
similar pattern (not shown). The larger RNA bands could
correspond to incompletely processed mRNA. This is
supported by the hybridization pattern obtained with
intronic probes (Fig. 1B). In addition to the larger tran-
scripts, these probes revealed faint bands (transcripts K3k
and K3e, Fig. 3) in the 1.4–1.65 kb range, plus the 0.9 kb
band (transcript K3f) previously detected with the exonic
probe. Thus, retention of multiple intronic sequences occurs
in prostate tissue. In contrast, several bands shorter than the
major mRNA were not detected with the intron-specific
probes. This suggests that their varying lengths arise from
alternative splicing or from the use of different poly(A)
signals which shorten the exons. Sequence analysis of the
K3b and K3 h transcripts (Fig. 3) supports this interpret-
ation. The expression of hKLK3 in tissues and in LNCaP
cell line differs in two major points. No short processed
transcripts were found in the LNCaP cells (not shown) and

the transcripts in the 1.9–2.1 kb range were less abundant
(Fig. 1C).
RNAs were purified from LNCaP polyribosomes to
analyze the association of hKLK3 transcripts with ribo-
somal and nonribosomal fractions, corresponding to trans-
lationally active and inactive mRNA, respectively. As
shown in Fig. 2, the major transcript (K3a) encoding
hK3/PSA was mainly associated with fractions containing
polyribosomes. Similar distribution was found for the
Fig. 1. Expression of the hKLK3 gene in prostate tissue (A and B) and in
LNCaP cells (C). Total RNA was analyzed by Northern blotting and
hybridized with probes derived from exon 2 (A), or from introns 1, 3 or
4 (B and C). Autoradiography was performed for 4 h (A) or 4 days
(B and C). The positions of the probes used are given in Fig. 3. The
sizes of the bands are indicated. The correspondence between the
bands and the cloned transcripts (lower case) was based on the length
of these transcripts determined by molecular sequencing (Fig. 3), plus
a poly(A) tail of about 200–250 bp and on their ability or not to
hybridize with the probes.
708 N. Heuze
´
-Vourc’h et al. (Eur. J. Biochem. 270) Ó FEBS 2003
transcripts corresponding to the 0.9 (transcript K3f), 1.65
(transcript K3e; not shown), 2.1 (transcript K3c) and 3.1 kb
bands, suggesting that these mRNA are efficiently transla-
ted in LNCaP cells. In contrast, the transcripts larger than
3.1 kb were mainly detected in the low density fractions
containing free, monosomal and small polysomal RNA and
would be thus poorly translated.
Structure of hKLK3 transcripts in the prostate

As the molecular cloning of the major transcript (K3a,
Fig. 3) encoding PSA, various alternative hKLK3 mRNAs
have been described [15,16,18–20]. Figure 3 shows their
schematic structure. The K3c-d transcripts retain part of the
intron 4 while the K3e-f transcripts retain the intron 3
[15,16,18]. In 2000, Tanaka et al. [19] described a partial
copy of a new hKLK3 transcript (K3g) with an alternative
splicing site at the beginning of the exon 3. We obtained the
3¢ lacking part of this mRNA by 3¢ RACE-PCR. As shown
in Fig. 3, the 3¢ end of this transcript (K3g) was identical to
the 3¢ end of the major transcript (K3a). Finally, two
transcripts with intronic sequences adjacent to the first exon
were recently described [20]. The former one is a transcript
containing the entire sequence of intron 1 (K3j, Fig. 3)
while the second one derived from an alternative splicing
within intron 1 (K3k). We amplified hKLK3 cDNAs by
RACE-PCR to examine the structure of short processed
transcripts. PCR products were fractionated on an agarose
gel then cloned. The clones YC140405.00, YC171105.00
Fig. 2. Polysomal distribution of the hKLK3 transcripts. Polysomes
were fractionated on a sucrose gradient. Aliquots (20 lg) of total RNA
from each fraction were hybridized to a probe derived from exon 3;
autoradiography was performed for 6 days. (T) Total RNA from
prostate tissue. The bands corresponding to cloned transcripts (lower
case) were arrowed.
K3-Ex1
K
3
-
sp5

K
3
-P
C
R2
SSI
K
3
-1
.5
K3-1.5
K
3
-
5055
K3-0.7rev2
K3-PCR2
K
3
-P
C
R2
K
3
-P
C
R2
K3-MU1
hK3/PSA
hK3/PSA

PSA-Tr
PSA-LM
PSA-LM
PSA-RP4
PSA-RP3
PSA-RP2
PSA-RP2
PSA-RP1
PSA-RP1
PSA-RP5
aa
69
261
238
180
218
220
104
227
a
b
(1)
k
j
l
(6)
h
(3)
g
(2)

f
e
d
c
i
(4)
nt
1460
860
1
90
2
1701
1627
709
1
3
2
0
850
>1040
> 583
>
1
9
4
5
> 1130
1000 bp
START

Ser189
Intron-2 probe
Exon-2 probe
His 41
Exon-3 probe
Asp 96
Intron-3 probe
Intron-4 probeIntron-1 probe
2
4
3
1
Fig. 3. Compilation of the hKLK3 transcripts. Intron numbers and position of the DNA probes used for hybridization are given in the genomic
DNA (grey). The variants (a to l) were classified according to their encoded protein (PSA to PSA-Tr). Numbers in exponent denote the new or
earlier described variants for which new data are given in the text. The length in nucleotides (nt) of the cloned sequences, without the poly(A) tail, is
shown at the left of the figure while the amino-acid (aa) number of the predicted prepro proteins is mentioned at the right. Exons are shown by boxes
and introns by the connecting lines, the lacking sequences of some transcripts are mentioned by dotted lines. Filled boxes represent the coding
sequences. Arrows in shaded boxes correspond to the position and direction of PCR primers used in the expression experiment. The positions of the
codons corresponding to the residues of the catalytic triad are indicated.
Ó FEBS 2003 Complex splicing of hKLK3 (Eur. J. Biochem. 270) 709
and YC100405 corresponded to 3 novel variants. The only
difference between the YC140405.00 sequence (transcript
K3b, accession no AJ459783; Fig. 3) and the major mRNA
(K3a) was the length of the 3¢ untranslated sequence. This
sequence was 586 nucleotides shorter in the K3b transcript.
Sequence analysis of the variant K3h corresponding to
the clone YC171105.00 revealed an additional intron
inside exon 3 (accession no AJ459782, Fig. 3). The clone
YC100405 was a partial copy of a new variant (K3i,
accession no AJ512346) retaining intron 4. Another partial

copy of a new alternative transcript was identified by
screening of an EST database with the hKLK3 genomic
sequence. This new transcript retained intron 2 sequences
(K3l, Fig. 3, accession no BE840537).
Expression of alternatively spliced hKLK3 transcripts
To determine whether alternatively spliced transcripts are
expressed in normal and pathological conditions, RT-PCR
was performed using total RNA from normal, BPH and
cancer specimens (Fig. 4). PCR primers were designed from
distant constitutive or alternative exons (Table 1 and Fig. 3)
and led to amplification of different size products from the
targeted transcripts and other putative transcripts with
intervening sequences. All PCRs performed on each tissue
specimen gave a major product, which displayed both the
expected size (Fig. 4, Table 1) and DNA sequence (not
shown). Additional faint bands were also observed, sug-
gesting amplification of longer transcripts containing inter-
vening sequences. This experiment indicates that all the
splicing isoforms tested are expressed in normal, BPH and
cancerous prostate tissues. However, it was not possible to
determine whether the malignant transformation alters the
production of alternatively spliced transcripts, as the
method used was not quantitative.
Fig. 4. Multiple alternative transcripts in the human prostate. Total
RNA from normal prostate (N), BPH or cancer was reverse-tran-
scribed. cDNAs were amplified by PCR using the primers given in
Table 1. The resulting PCR products were separated on agarose gel
and visualized by ethidium bromide. C: control without cDNA. From
0.1 to 1 kb, the increment of the DNA ladder was 100 bp.
Table 2. Exon-intron boundaries of the hKLK3 gene. Exon and intron numbers refer to the numbers given in Fig. 3. Letters at exon or intron

numbers indicate the variant exon or intron found in the referred transcript while the (¢) symbol indicates an additional exon or intron. Exon
sequences are in uppercase and introns in lowercase. Residues that are identical with the consensus sequences are in bold or underlined. M ¼ Aor
C, Y ¼ CorU,R¼ AorG,N¼ any.
Exon No.
(transcript)
Size
(bp)
Intron
No.
Size
(bp)
5¢ donor seq.
MAGguragu
Branch site
ynyuray
3¢ acceptor seq.
(y
(n)
ryagG)
1 (K3a-h, l) 87 1 1239 UUGgugaga cccugau cccccucugcagGCG
1j (K3j) 1485 2 1639 GAAgugagu uccucau cuuccuccccagCAA
1 k (K3k) 510 1k 815 GCUgugagu cccugau cccccucugcagGCG
2 (K3a-f, h- k) 160 2 1639 GAAgugagu uccucau cuuccuccccagCAA
2 (K3g) 160 2 g 1768 GAAgugagu uccugaa auuccu cagGCC
3 (K3a-d,i- k) 287 3 143 AGUguacgc ccacaac cccguagUCU
3 g (K3g) 248 3 143 AGUguacgc ccacaac cccguagUCU
3 h (K3h) 147 3¢ 123 CAGccacga cacuggggac ggggcagCAU
3’h (K3h) 17 3 143 AGUguacgc ccacaac cccguagUCU
4 (K3ab,e,gh,jk) 137 4 1375 UGCgugagu gacugac cccuuagGGU
4 (K3cd) 137 4c 933 UGCgugagu ccuccac cccacagUGG

Poly(A) signal
AAUAAA
Poly(A) cleavage site
Ca n
(< 10)…
yguguuyy
Ca n
(< 10)…
u rich
5 (K3a,e,g) 794
AAUAAA Gu ugugugac
5b 207 AGUAAA Caggccaagacucaag
5c 1236 AAUAAA Gu ugugugac
5d 1034 CAUAAC Gu ugugugac
3f (K3f) 465 AAGAAA Cc uguuauuu
5 h 306 AAGAAA Ca aguguuuc
4i >521
5 (K3j,k) >36
710 N. Heuze
´
-Vourc’h et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Exon/intron structure analysis
We examined the intron/exon boundaries of the hKLK3 gene
to see if the sequence signals required for premRNA splicing
was preserved (Table 2). While the AG dinucleotide imme-
diately preceding the exon was always present in the hKLK3
acceptor sequences, one donor sequence (intron 3¢,K3h)
lacked the well-conserved GU dinucleotide. There were also
several mismatches in the exonic part of the consensus, with
great base variations at each position. Analysis of the hKLK3

gene using a search algorithm (itfly.org/
seq_tools/splice.html) revealed 21 potential donor and 36
potential acceptor sequences (including the sites for splicing
of introns 1, 2 and 4). One found 13 nucleotides upstream of
the variant donor site of intron 3¢, was a canonical 5¢ splice
site. This algorithm did not detect the alternative acceptor
sequence used for transcripts k3c-d and, the real sites defining
the boundaries of introns 3 and 3¢. This suggests that the
splice sites are not optimal, a property often found for
retained introns [21]. Putative branch sites with adjacent
polypyrimidine tracts were found 20–30 nucleotides
upstream of all the acceptor sites (Table 2).
It has been known for some time that many alternatively
spliced exons, small exons or exons with weak splice sites
rely upon the activity of enhancers for their inclusion in
mRNA [22]. As several splicing events affect the region
surrounding intron 3, we searched for putative regulatory
signals (Fig. 5). Intron 3 is studded with G triplets and
quadruplets. It has been suggested that G triplets enhance
splicing efficiency and help to determine exon–intron
borders [23,24]. Two G triplets and one G quadruplet
belong to a 22 nucleotide duplicated element that we termed
pISE (putative intron splicing enhancer). Each pISE copy
(Fig. 5) also contains two short sequences, GGGUCUG
and GAGGA, related to known splicing enhancers [25,26].
The first short sequence is similar to the consensus
GGGGCUG of the intron splicing enhancer found down-
stream of the microexon of the chicken cardiac troponin T
gene. In this gene, the enhancer binds the bridging splicing
factor SF1 and increases recognition of the upstream

microexon of 7 nucleotides [25]. There is also an alternative
microexon of 17 nucleotides upstream of the pISE in
hKLK3. The GAGGA motif is present in intron 3 and in the
17 nucleotide microexon. In the latter, it lies downstream of
a sequence motif similar to the (U)GGACCNG consensus
sequence of an exonic splicing enhancer [26]. Another
upstream sequence (UGGACCUG) fits the same consensus
motif. Two other exonic enhancer sequence motifs
(UCCUC and CCACCC) previously identified by in vitro
selection of randomized RNA sequences [27] were found in
exon 3.
Structure of hKLK3 proteins
The predicted amino-acid sequences of proteins encoded by
the alternatively spliced mRNAs are shown in Fig. 6. The
<−−−−−− PISE −−−−> <−−−−−− PISE −−−−>
Fig. 5. Sequences of the region surrounding intron 3. The sequences of several transcripts were aligned with the premRNA sequence derived from
the hKLK3 gene sequence. The dotted lines correspond to the intervening sequences. The putative regulatory signals are indicated in colour. The
dinucleotide of the donor (red) and acceptor (green) splice site signals are highlighted, as are the putative branch points (grey). Nucleotides of the
polypyrimidine tracts are in red. The G stretches are highlighted in yellow while the nucleotide sequences of putative splicing enhancers are in blue.
Ó FEBS 2003 Complex splicing of hKLK3 (Eur. J. Biochem. 270) 711
conservation of the N-terminal part of PSA, including the
scretion signal peptide and the propeptide, suggests that all
the PSA-RPs (PSA-related proteins) were synthesized as
prepro proteins. While PSA-RP1, PSA-RP2 and PSA-RP5
differ from PSA at the C-terminal region. PSA-RP3 and
PSA-RP4 are shorter than PSA due to in frame deletions. In
PSA-RP3, the deletion results in the loss of asparagine-45
that is the binding site for the carbohydrate chain in PSA
[19]. Forty-two amino acids, including one cysteine residue
and the aspartate residue-96 of the catalytic triad, are

deleted in PSA-RP4. The K3l transcript (from the EST
database) contains a premature stop codon located at the
beginning of the retained intron 2. It might encode a
truncated form of prepro PSA (PSA-Tr, PSA-truncated).
The transcripts K3j and k encode a protein (PSA-LM [20]),
sharing only the signal peptide with PSA due to the creation
of a novel ORF by the retention of intron 1 sequences.
Although recombinant PSA-RP2 has been produced in a
heterologous eukaryotic cell system [18], there has been no
report on expression of this variant in prostate. Therefore,
polyclonal antibodies were raised against a peptide corres-
ponding to the C-terminal sequence of PSA-RP2. As shown
in Fig. 7, these antibodies recognized recombinant PSA-
RP2 but not PSA purified from seminal fluid. Moreover, a
protein with a molecular mass similar to that of recombi-
nant PSA-RP2 was detected in a protein extract from a
cancerous prostate tissue (Fig. 7), revealing production of
PSA-RP2 in vivo.
Discussion
We have used Northern blotting, molecular cloning and a
database search to show that the hKLK3 gene produces at
least 15 transcripts, of 0.7 to 6.1 kb, in prostate. Thus, the
expression and splicing of the hKLK3 gene is more complex
than previously thought [17]. All transcripts larger than the
major mRNA encoding hK3/PSA contain intronic
sequences. Their polysomal distribution indicates that the
2.1 (K3c, PSA-RP1) and 3.1 kb transcripts are mature
mRNAs efficiently translated in LNCaP cells, whereas the
largest transcripts seem to be weakly translated. As the large
hKLK3 transcripts retaining introns were detected in the

cytosolic fraction, it is unlikely that they are splicing
intermediates. These transcripts might be either aberrant
(poorly spliced with nonsense codon) or coding transcripts.
The presence of a premature stop codon in the k3l transcript
corresponding to PSA-Tr supports the hypothesis of
aberrant hKLK3 transcripts. Degradation of aberrant
transcripts is thought to occur in the cytoplasm via the
mRNA surveillance system that depends upon translation
[28–30]. This could explain both cytoplasmic localization
and association with ribosomes of the poorly spliced hKLK3
transcripts. Further investigations are required to determine
whether aberrant hKLK3 transcripts significantly accumu-
late before degradation. Alternatively, the larger ones could
be coding transcripts. An unusual feature of the hKLK3
gene is that the open reading frame continues in intron 1
resulting in the PSA-LM protein [20]. As the large
transcripts hybridized with the intron 1 probe, they might
encode PSA-LM. In this case, weak association of the large
hKLK3 transcripts with polysomes could be due to peculiar
structures that reduce translation efficiency [31]. Numerous
cis-acting sequences and trans-acting cytoplasmic proteins
participating in mRNA stability, localization or translation,
PSA MWVPVVFLTLSVTWIGA APLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIR NKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHD
PSA-RP1
MWVPVVFLTLSVTWIGA APLILSR IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIR NKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSH D
PSA-RP2
MWVPVVFLTLSVTWIGA APLILSR IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIR NKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSH D
PSA-RP3
MWVPVVFLTLSVTWIGA APLILSR IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIR KPGDDSSH D
PSA-RP4

MWVPVVFLTLSVTWIGA APLILSR IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIR NKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSS
PSA-RP5
MWVPVVFLTLSVTWIGA APLILSR IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIR NKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSH D
PSA-Tr
MWVPVVFLTLSVTWIGA APLILSR IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIR K
PSA-LM
MWVPVVFLTLSVTWIG ERGHGWGDAGEGASPDCQAEALSPPTQHPSPDRELGSFLSLPAPLQAHTPSPSILQQSSLPHQVPAPSHLPQNFLPIAQPAPCSQLLY
PSA LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGW GSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGD
SGGPLVCNGVLQGITSWGSEPCALPERP
PSA-RP1 LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCS
WVILITELTMPALPMVLHGSLVPWRGGV
PSA-RP2 LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEE
CTPGPDGAAGSPDAWV
PSA-RP3 LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGD
SGGPLVCNGVLQGITSWGSEPCALPERP
PSA-RP4 IEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGD
SGGPLVCNGVLQGITSWGSEPCALPERP
PSA-RP5 LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCS
VSHPYSQDLEGKGEWGP
PSA SLYTKVVHYRKWIKDTIVANP
PSA-RP3 SLYTKVVHYRKWIKDTIVANP
PSA-RP4 SLYTKVVHYRKWIKDTIVANP
Fig. 6. Alignment of the predicted hKLK3 proteins. The signal peptide is highlighted in yellow and the propeptide in blue. The amino-acid residues of
the catalytic triad are in red, while the binding site for the carbohydrate chain is in green. Sequences divergent to the PSA sequence are highlighted in
grey.
Fig. 7. Detection of PSA-RP2 in prostate tissue. PSA from seminal
fluid (40 ng), cytosolic proteins from CHO cells expressing recom-
binant PSA-RP2 (60 lg) and from a cancerous prostate tissue (250 lg)
were subjected to SDS/PAGE and analyzed by Western blot using
the polyclonal anti-RP2 Ig. The band corresponding to PSA-RP2 is

indicatedbyanarrow.
712 N. Heuze
´
-Vourc’h et al. (Eur. J. Biochem. 270) Ó FEBS 2003
have been identified in eukaryotes. A search for cis elements
within the hKLK3 gene sequence using the UTRScan
computer program [32] revealed no conserved sequences
involved in (de)stabilizing, locating or translating mRNAs.
However, unconserved cis-acting sequences could play a
regulatory role in the translation efficiency of the large
hKLK3 transcripts.
Our study reveals the large 3¢-UTR diversity of hKLK3
transcripts. Two well documented functions of 3¢-UTRs are
mRNA stabilization and its localization in specific regions
of the cytoplasm [32]. The use of different polyadenylation
sites suggests that there is a post-transcriptional regulation
of hKLK3 gene expression. This is supported by data
indicating that the 3.1 kb transcript is more unstable than
the major hKLK3 mRNA [33]. Functional analyses will be
needed to assess the role of the 3¢UTR in the stability of
hKLK3 mRNAs in normal and pathological prostatic cells.
The process by which constitutive and alternative exons
are recognized in a premRNA is complex. The early steps
of spliceosome assembly involve recognition of consensus
elements at both ends of the intron. Although these
sequences are usually short, they are often degenerate.
Nevertheless, about 99% of splice site pairs are GT-AG
[34]. The alternative intron 3¢ of hKLK3 does not follow
this rule, but has unusual CC-AG pairs. Recognition of
this atypical site is probably related to the presence of a

canonical site upstream to the variant. Indeed, Burset et al.
suggested that uncanonical sites could function exclusively
in association with a canonical site [34]. In the other cases,
reasonably conserved signals were found at both ends of
the hKLK3 introns; however, their relative strength
remains to be determined. It is clear that conserved
sequences near the 3¢ and 5¢ splice sites are generally
insufficient for selecting true splice sites among abundance
of similar sequences. Unconserved sequences commonly
named splicing enhancers and silencers provide more
information to specific regulatory factors that interact or
interfere with the splicing machinery. We looked for
putative regulatory sequences because of the complexity of
the splicing events affecting the middle of the hKLK3 gene.
Intron 3 contains a high concentration of G triplets; these
are frequently found close to 5¢ splice sites in mammals
[23,24]. This well-established splicing enhancer promotes
the selection of a 5¢ splice site by recruiting U1 snRNP.
Many other putative splicing enhancers were detected in
the alternative exons and introns, suggesting that there is
considerable information in the various segments of the
hKLK3 premRNA. Some sequences also contain overlap-
ping elements. We identified a 22-nucleotide repeat (pISE)
which contains G triplets and an internal motif known to
recruit SF1. Thus, pISE could be involved in the
determination of exon-intron borders via interaction of
the G sequences with U1 snRNPs and, in definition of the
microexon 3¢ via recruitment of SF1 by the internal motif
[25]. These observations suggest that the complex splicing
of hKLK3 probably reflects the probability of occupancy

of individual sites and the cross-talk between multiple
interactions, as in other genes [35]. The splicings result in
two short introns (3 and 3¢) and a 17 nucleotide
microexon. This is unusual as the exons are typically
100–200 nucleotides in human, and the introns are much
longer, averaging about 3 kb. Only about 10% of the
introns are classified as short (< 134 nucleotides), while no
more than 4% of vertebrate internal exons are shorter than
50 nucleotides [36].
To date, 12 hKLK3 transcripts have been cloned and
sequenced. The proteins predicted from the nucleotide
sequences are PSA, truncated PSA and six alternate
proteins. Five predicted proteins are PSA variants (PSA-
RP1 to RP5) that could be synthesized as precursors. The
presence of a common signal peptide suggests that all these
PSA-RPs are secreted from prostate cells. Previous recom-
binant experiments [15,18] and the identification of PSA-
RP1 in the spent medium of LNCaP cells [37,38] strongly
support this assertion. In the present time, two PSA-RPs
have been identified in prostate tissue, PSA-RP1 [37] and
PSA-RP2 using immunohistochemical and Western blot
analysis, respectively. Characterization of other PSA-RP
variants is currently under investigation. The variation in
the mRNA will result in several great changes in the amino-
acid sequences that probably interfere with the protease
activity of hK3/PSA. As PSA function depends on this
activity, we need to know how these variants that seem to
have no enzymatic activity, influence prostate physiology
and pathology. By contrast to the PSA-RPs, the protein
PSA-LM encoded by the transcripts containing intron 1 is

quite unlike PSA. A recombinant form of this protein has
been recently characterized [20]. PSA-LM has also been
found in the secretory epithelial cells of prostate; however,
its function remains unknown. All these observations
emphasize the complexity of the protein resulting from
hKLK3 gene expression.
Numerous efforts are made to ameliorate the diagnostic
value of the PSA assay. The major aim in this field is to
enhance the discrimination of patients with BPH from those
with Pca. One way would be to use additional markers. As
cancer is said to alter the splicing pattern of some genes [39],
some variants of PSA could be useful to improve the tumor
selectivity of the PSA assay. Additional studies are required
to determine the clinical values of these PSA variants.
Acknowledgements
We are indebted to Drs Lanson and Haillot of the Department of
Urology, Hoˆ pital Bretonneau de Tours for providing human prostate
tissues. We thank Mme E. Bataille
´
, Drs Gutman and Rosinski-Chupin
for their assistance and O. Parkes for critically reviewing this manu-
script before its submission. This work was supported by grants from
the Association pour la Recherche sur le Cancer, the Ligue contre le
Cancer (Comite
´
d’Indre-et-Loire) and from the Association de
Recherche sur les Tumeurs de la Prostate.
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