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 codingfor the tumor marker PSA prostate-specific-antigen Nathalie Heuze´-Vourc’h, Vale´rie Leblond and Yves Courty Laboratoire d’Enzymologi

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)1do

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 hKLK3gene 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 mM glutamine in the presence of the synthetic androgen R1881 (0.1 nM; 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: courty@univ-tours.fr

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)

Polysomal RNA preparation

LNCaP cells (5· 108) 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 mM Tris/HCl pH 8.0,

250 mM KCl, 5 mM MgCl2) containing 250 mM sucrose,

2 mMdithiothreitol, 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 2C The supernatant was

supple-mented with 2% Triton X100 and 0.2 mgÆmL)1of 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 2C in an L5

ultracentrifuge (Beckman) equipped with an SW41 rotor

Samples of 500 lL were collected from the sucrose gradient,

and 250 mM 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 H2O 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 [a32P]-labeled probe using the Klenow fragment of

Escherichia coliDNA polymerase I After heat

denatura-tion, the biotinylated unlabeled strand was captured using

Streptavidin MagneSphere 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 94C for 2 min and

30 cycles of 94C for 30 s, annealing temperature (Table 1) for 30 s and 75C for 1.5 min The resulting fragments were purified using the Wizard PCR preps DNA purifi-cation system (Promega Corp.) and 50–100 ng were labeled with [a32P]dCTP by random priming

Northern blot hybridization was performed overnight at

68C with the QuikHyb Hybridization solution (Strata-gene, La Jolla, CA, USA) Blots were washed at 68C for

2· 30 min in 2 · 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 94C for 2 min; 5 cycles of denaturation at 94 C for

30 s, annealing and elongation at 72C for 3.30 min; 5 cycles of denaturation at 94C for 30 s, annealing and elongation at 70C for 3.30 min; 25 cycles, 94 C for 30 s,

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

RT-PCR

68C 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¢- and a 3¢-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: 94C for 3 min and 35

cycles at 94C for 30 s, 68 C or 55 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 nonriboribo-somal 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 hKLK3gene 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.

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 hKLK3transcripts 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-PCR2

K3-1.5

K3-5055

K3-0.7rev2 K3-PCR2

K3-PCR2

K3-PCR2

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

1902

1701

1627

709

1320

850

>1040

> 583

> 1945

> 1130

1000 bp

START

Ser189

Intron-2 probe

Exon-2 probe His 41

Exon-3 probe

Asp 96

Intron-3 probe Intron-4 probe Intron-1 probe

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.

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 ESTdatabase 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 hKLK3gene 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 A or

C, Y ¼ C or U, R ¼ A or G, N ¼ any.

Exon No.

(transcript)

Size (bp)

Intron No.

Size (bp)

5¢ donor seq.

MAGguragu

Branch site ynyuray

3¢ acceptor seq.

Poly(A) signal AAUAAA

Poly(A) cleavage site

Ca n(< 10)…yguguuyy

Ca n(< 10)… u rich

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¢, K3 h)

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 (http://www.fruitfly.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

AUGAGCCUCCUGAAGAAUCGAUUCCUCAGGCCAGGUGAUGACUCCAG -Pre UGAAGGUCA UGGACCUG C CCACCC AGGAGCCAGCACUGGGGAC C CCU G CU A G CCUC AGG CU GGGG C AGCAU UGAACCAG AGGAGUGUACGCCUGGGCC

-CAUUGAACCAGAGGAGU -<−−−−−− PISE −−−−> -CAUUGAACCAGAGGAGU -<−−−−−− 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.

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 hKLK3transcripts 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 hKLK3transcripts 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 MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHD PSA-RP1 MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHD PSA-RP2 MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHD PSA-RP3 MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIR -KPGDDSSHD PSA-RP4 MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSS PSA-RP5 MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHD PSA-Tr MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRK

PSA-LM MWVPVVFLTLSVTWIGERGHGWGDAGEGASPDCQAEALSPPTQHPSPDRELGSFLSLPAPLQAHTPSPSILQQSSLPHQVPAPSHLPQNFLPIAQPAPCSQLLY

PSA LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERP PSA-RP1 LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSWVILITELTMPALPMVLHGSLVPWRGGV PSA-RP2 LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEECTPGPDGAAGSPDAWV PSA-RP3 LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERP PSA-RP4 -IEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERP PSA-RP5 LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSVSHPYSQDLEGKGEWGP

PSA SLYTKVVHYRKWIKDTIVANP

PSA-RP3 SLYTKVVHYRKWIKDTIVANP

PSA-RP4 SLYTKVVHYRKWIKDTIVANP

Fig 6 Alignment of the predicted hKLK3proteins 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 indicated by an arrow.

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

hKLK3transcripts

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

hKLK3mRNAs 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

hKLK3premRNA 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 hKLK3gene 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, Hoˆpital 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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