Human SHBG mRNA translation is modulated by alternative 5'-non-coding exons 1A and 1B.
ABSTRACT The human sex hormone-binding globulin (SHBG) gene comprises at least 6 different transcription units (TU-1, -1A, -1B, -1C, -1D and -1E), and is regulated by no less than 6 different promoters. The best characterized are TU-1 and TU-1A: TU-1 is responsible for producing plasma SHBG, while TU-1A is transcribed and translated in the testis. Transcription of the recently described TU-1B, -1C, and -1D has been demonstrated in human prostate tissue and prostate cancer cell lines, as well as in other human cell lines such as HeLa, HepG2, HeK 293, CW 9019 and imr 32. However, there are no reported data demonstrating their translation. In the present study, we aimed to determine whether TU-1A and TU-1B are indeed translated in the human prostate and whether 5' UTR exons 1A and 1B differently regulate SHBG translation.
Cis-regulatory elements that could potentially regulate translation were identified within the 5'UTRs of SHBG TU-1A and TU-1B. Although full-length SHBG TU-1A and TU-1B mRNAs were present in prostate cancer cell lines, the endogenous SHBG protein was not detected by western blot in any of them. LNCaP prostate cancer cells transfected with several SHBG constructs containing exons 2 to 8 but lacking the 5'UTR sequence did show SHBG translation, whereas inclusion of the 5'UTR sequences of either exon 1A or 1B caused a dramatic decrease in SHBG protein levels. The molecular weight of SHBG did not vary between cells transfected with constructs with or without the 5'UTR sequence, thus confirming that the first in-frame ATG of exon 2 is the translation start site of TU-1A and TU-1B.
The use of alternative SHBG first exons 1A and 1B differentially inhibits translation from the ATG situated in exon 2, which codes for methionine 30 of transcripts that begin with the exon 1 sequence.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Salivary adenoid cystic carcinoma (SACC) is a common type of salivary gland cancer. The poor long-term prognosis of patients with SACC is primarily due to local recurrence, distant metastasis and perineural invasion. MicroRNAs (miRNAs) have been identified as important post-transcriptional regulators, which are involved in various biological processes. The aim of the present study was to identify the miRNA expression profiles that are involved in the metastatic progression of SACC. Therefore, microarray technology was employed to identify miRNA expression profiles in an SACC cell line, ACC-2, and a highly metastatic SACC cell line, ACC-M, which was screened from ACC-2 by a combination of in vivo selection and cloning in vitro. Differences in miRNA expression were assessed by quantitative polymerase chain reaction (qPCR) assay. In addition, the potential target genes that are regulated by selected miRNAs were analyzed by various target prediction tools. The microarray data revealed that the levels of 38 miRNAs significantly differed between the ACC-M cells and the control ACC-2 cells. Six miRNAs (miR-4487, -4430, -486-3p, -5191, -3131 and -211-3p) were selected to validate the microarray data via qPCR. The expression of two miRNAs (miR-4487 and -4430) was significantly upregulated in the ACC-M cells, while the expression of two other miRNAs (miR-5191 and -3131) was significantly downregulated in the ACC-M cells. The potential target genes that were identified to be controlled by the six selected miRNAs were divided into four groups according to function, as follows: Apoptosis and proliferation (46 genes), cell cycle (30 genes), DNA damage and repair (24 genes) and signaling pathway (30 genes). The identification of microRNA expression profiles in highly metastatic SACC cells may provide an improved understanding of the mechanisms involved in metastatic progression, which would aid in the development of novel strategies for the treatment of SACC.Oncology letters 06/2014; 7(6):2029-2034. · 0.24 Impact Factor
Human SHBG mRNA Translation Is Modulated by
Alternative 59-Non-Coding Exons 1A and 1B
Toma `s Pino ´s1,2, Anna Barbosa-Desongles1, Antoni Hurtado1, Albert Santamaria-Martı ´nez1, Ine ´s de
Torres3, Jaume Revento ´s1, Francina Munell1*
1Institut de Recerca Hospital Universitari Vall d’Hebro ´n, Barcelona, Spain, 2Centro de Investigacio ´n Biome ´dica en Red de Enfermedades Raras (CIBERER), Valencia, Spain,
3Servei d’Anatomı ´a Patolo `gica, Hospital Universitari Vall d’Hebro ´n, Barcelona, Spain
Background: The human sex hormone-binding globulin (SHBG) gene comprises at least 6 different transcription units (TU-1,
-1A, -1B, -1C, -1D and -1E), and is regulated by no less than 6 different promoters. The best characterized are TU-1 and TU-
1A: TU-1 is responsible for producing plasma SHBG, while TU-1A is transcribed and translated in the testis. Transcription of
the recently described TU-1B, -1C, and -1D has been demonstrated in human prostate tissue and prostate cancer cell lines,
as well as in other human cell lines such as HeLa, HepG2, HeK 293, CW 9019 and imr 32. However, there are no reported data
demonstrating their translation. In the present study, we aimed to determine whether TU-1A and TU-1B are indeed
translated in the human prostate and whether 59 UTR exons 1A and 1B differently regulate SHBG translation.
Results: Cis-regulatory elements that could potentially regulate translation were identified within the 59UTRs of SHBG TU-1A
and TU–1B. Although full-length SHBG TU-1A and TU-1B mRNAs were present in prostate cancer cell lines, the endogenous
SHBG protein was not detected by western blot in any of them. LNCaP prostate cancer cells transfected with several SHBG
constructs containing exons 2 to 8 but lacking the 59UTR sequence did show SHBG translation, whereas inclusion of the
59UTR sequences of either exon 1A or 1B caused a dramatic decrease in SHBG protein levels. The molecular weight of SHBG
did not vary between cells transfected with constructs with or without the 59UTR sequence, thus confirming that the first in-
frame ATG of exon 2 is the translation start site of TU-1A and TU-1B.
Conclusions: The use of alternative SHBG first exons 1A and 1B differentially inhibits translation from the ATG situated in
exon 2, which codes for methionine 30 of transcripts that begin with the exon 1 sequence.
Citation: Pino ´s T, Barbosa-Desongles A, Hurtado A, Santamaria-Martı ´nez A, de Torres I, et al. (2010) Human SHBG mRNA Translation Is Modulated by Alternative
59-Non-Coding Exons 1A and 1B. PLoS ONE 5(11): e13844. doi:10.1371/journal.pone.0013844
Editor: Alfons Navarro, University of Barcelona, Spain
Received June 11, 2010; Accepted October 6, 2010; Published November 4, 2010
Copyright: ? 2010 Pino ´s et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Ministerio de Sanidad y Consumo (PI052684), Fundacio ´n para la Investigacio ´n en Urologı ´a. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Sex hormone-binding globulin (SHBG) is a dimeric glycopro-
tein that transports sex steroids in the blood and regulates their
access to target tissues . The human SHBG gene is located in
the short arm of chromosome 17 (17p13.1), contains at least 6
different transcription units, which are constituted of a common
region that spans exons 2 to 8, and 6 alternative first exons .
These exons are named 1, 1A, 1B, 1C, 1D and 1E, following their
59 to 39 orientation on the positive strand of chromosome 17, and
are all spliced to exon 2 using the same 39 splice site . Exons 1
and 1A (previously known as alternative exon 1) were the first to
be characterized and have been extensively studied [1,3,4,5,6,7,8].
Exon 1 encodes a signal peptide and is responsible for production
of plasma SHBG by the hepatocytes. TU-1 is regulated by
promoter 1 sequence that contains several binding sites for liver-
enriched transcription factors [8,9]. TU-1A begins with the exon
1A sequence, which does not contain an ATG in frame with the
SHBG coding sequence. It has been proposed that TU-1A
initiates translation at the first ATG in frame of exon 2, which
codes for methionine 30 of transcripts beginning with exon 1 [7,8].
It has also been described that TU-1A is regulated by an
alternative promoter sequence [6,8] that proved to be very active
when transfected in the GC2 mouse germ cell line . The
presence of full-length TU-1A has been demonstrated in human
testis, liver, prostate, breast, and brain tissue, in human cancer cell
lines derived from prostate (LNCaP) and breast (MCF-7), and in
the testis of mice containing the 11-kb human SHBG transgene
Exons 1B, 1C, 1D (also known as exon 1N) and 1E, have been
recently identified and described in human prostate tissue, in
LNCaP, PC3, and PZ-HPV7 prostate cancer cell lines, and in
several cancer cell lines originating in other tissues [2,5]. As
occurred with exon 1A, exons 1B, 1C, 1D and 1E do not contain
an ATG in frame with the SHBG coding sequence. Therefore, as
is the case of exon 1A, exons 1B, 1C, 1D, and 1E are 59
untranslated regions (59UTRs) of their corresponding TUs and
might also initiate translation at the first in-frame ATG of exon 2
. Full-length TU-1B transcripts have been detected in LNCaP
cells and in human prostate tissue , TU-1D in LNCaP and
PLoS ONE | www.plosone.org1November 2010 | Volume 5 | Issue 11 | e13844
MCF-7 cell lines , and TU-1C in the rhabdomyosarcoma CW
9019 and neuroblastoma imr 32 cell lines .
Alternative promoter usage has been shown to enable
diversified transcriptional regulation in different cellular conditions
or development stages [10,11], and along with alternative splicing
are the primary sources of 59UTR transcript diversity .
Estimates of the number of genes with alternative 59UTRs vary
from 12% to 22%, while those of alternative promoter usage range
from 10% to 18% . Recent studies have shown that 59UTRs
play an important role in regulation of gene expression in a variety
of organisms (microbes, plants, and animals) . 59UTR-
mediated regulation has been shown to modulate gene expression
through stimulatory and inhibitory mechanisms [13,14], influenc-
ing the mRNA secondary structure, mRNA stability and
translation efficiency [4,13,14,15]. Specifically, it has been shown
that occurrence of start codons and open reading frames upstream
of the authentic start codon (uAUGs and uORFs, respectively)
may affect mRNA translation [12,14,16,17]. The different SHBG
59UTRs exhibit many of the features associated with cellular
mRNAs, whose expression is tightly controlled at the level of
translation, including uORFs and thermodynamically stable
predicted RNA structures [2,5]. The present study determines
the impact of exon 1A and 1B 59UTRs on SHBG translation.
Identification of potential translation regulatory elements
in SHBG exons 1A and 1B
To investigate whether SHBG TU-1A and TU-1B are
translated and how their alternative first exons modulate SHBG
translation, we analyzed these sequences to identify potential
translation regulatory elements, such as uAUGs, G/C-rich
sequences, and stable secondary structures within 59UTRs, known
to serve as effective barriers to the scanning ribosomes [18,19,20].
SHBG TU-1 contains a short 59UTR of 79 nucleotides without
an uAUG (Genbank EU352659) [1,5]. In contrast, TU-1A has a
59UTR of 158 nucleotides with 2 uAUGs (one in the exon 1A
sequence and the other in the exon 2 sequence) (Genbank
X16351.1; Figure 1A), and TU-1B contains a 59UTR of 167
nucleotides with 1 uAUG (the one in the exon 2 sequence)
(Figure 1A). The uAUG of exon 1A produces an uORF of only 2
codons, whereas the uAUG found in exon 2 of both TU-1A and
TU-1B generates an uORF of 11 codons, and the corresponding
stop codon overlaps with the predicted translation start site of the
SHBG ORF (Figure 1B). Additionally, both the M-fold and P-fold
programs predict a higher level of thermodynamic stability for the
secondary structures of both TU-1A [dG=240.3 kcal/mol (M-
fold);  and dG=244.02 kcal/mol (P-fold), with 58.22% G/C]
and TU-1B [dG=263.8 kcal/mol (M-fold);  and dG=
269.9 kcal/mol (P-fold), with 67.9% G/C], in comparison with
TU-1 [dG=218.3 kcal/mol (M-fold) and dG=218 kcal/mol (P-
fold), with 58.2% G/C] (Figure 2A, B and Figure 3; Table 1).
According to the M-fold program, in the TU-1A 59UTR, the exon
2 translation start site is found at the end of a stable hairpin (HP,
dG=28.50 kcal/mol; Figure 2B), while in the case of the TU-1B
59UTR, it is found within a loop preceded by 4 minor hairpins
(HP 1, 2, 3, and 4; Figure 3), and one major hairpin (HP 5,
dG=226.20 kcal/mol; Figure 3).
Absence of SHBG protein translation in human prostate
In the present study, mRNAs corresponding to a region (Ex2-
Ex5) common to all SHBG TUs (Figure 4A), as well as exon 1A
and exon 1B full-length transcripts (Figure 4B, C) were detected by
RT-PCR. We then performed western blot using the SHBG
11F11 antibody (which recognizes the aminoacids encoded by
exon 7 and the beginning of exon 8; Figures S1, S2) in order to
detect the SHBG protein translated from these human prostate
mRNAs. Two bands were detected in human prostate tissue,
which were identical in size (52 and 48 kDa) to those seen in
plasma and testis (Figure 5A, B). However, when total protein
extracts from LNCaP, PC3, and PZ-HPV7 were analyzed, no
SHBG protein was detected, except in LNCaP cells transfected
with the Flag-EX2-EX8-SHBG construct, used as a positive control
of SHBG translation, (Figure 5D), in which a protein band
approximately 35 kDa in size was observed. This size matched
well with the predicted molecular weight of the 345 amino acids of
the SHBG protein fused to the 8 amino acids of the Flag tag
(Figure 5C). These results support the idea that the SHBG protein
is not present or at least cannot be detected by this technique in
LNCaP, PC3, and PZ-HPV-7 cells. We then checked the capacity
of these prostate cell lines to secrete SHBG, using the HepG2
hepatocarcinoma cell line as a positive control. Western blot
analysis of supernatants from the 4 cell lines grown without FBS
for 72 h demonstrated the protein exclusively in HepG2 culture
medium, and showed a molecular size (52 and 48 kDa) identical to
that of the protein found in plasma (Figure 5E). The absence of
SHBG protein in prostate cell lines suggested that the protein
found in prostate human tissue corresponded to plasma SHBG.
Deglycosylation of SHBG extracted from prostate tissues reduced
both bands to a lower molecular size of approximately 37 kDa
(data not shown), confirming that both bands corresponded to the
SHBG monomers characteristic of the secreted protein and were
likely of blood origin.
59 UTR sequences modulate SHBG gene expression
To test whether absence of the SHBG protein in prostate
cancer cell lines resulted from reduced translation due to a
modulatory action of the 59UTR sequences of exon 1A and 1B
on the translation efficiency from the first ATG in frame of exon
2, LNCaP cells were transfected with several SHBG constructs
with and without exon 1A and exon 1B 59UTR sequences
When LNCaP cells were transfected with the SHBGEX2-EX8-
pDsRed construct, which did not include any SHBG 59UTR
sequence, an immunoreactive band of approximately 62 kDa in
size was detected. This band matched well with the predicted
molecular weight of the SHBG-pDsRed fusion protein (345 aa
from the SHBG protein plus 225 aa from the C-terminus pDsRed-
fused tag) (Figure 5C). Again, no band was detected in LNCaP
non-transfected cells, while the 35 kDa band was observed when
these cells were transfected with the Flag-EX2-EX8-SHBG construct
(Figure 5F). Additionally, when LNCaP cells were transfected with
the tag-less Ex2-Ex8-pCDNA 3.1 construct (without any SHBG
59UTR sequence), a band of approximately 35 kDa was detected.
Therefore, the SHBG protein is only detected in LNCaP
transfected cells, and the differing molecular weight of the
immunoreactive bands was caused by the presence and length of
the tags fused to the SHBG protein, which is an additional
indicator of the specificity of the antibody.
We next determined the influence of exons 1A and 1B on
SHBG translation. Western blot analysis of LNCaP cells
transfected with the SHBGEX1A/1B-EX8-pDsRed constructs, which
include the exon 1A or 1B 59UTRs, showed that the amount of
SHBG protein was much lower than in cells transfected with the
SHBGEX2-EX8-pDsRed construct, which did not present any
59UTRs (Figure 6A, B). Specifically, almost no SHBG was
detected in cells transfected with the SHBG1B-EX8-pDsRed
construct (Figure 6A). The similar amounts of SHBG mRNA in
SHBG Regulation by 59 UTRs
PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e13844
all LNCaP cells transfected with the different SHBG-pDsRed
(Figure 6A), rules out the possibility that variations in SHBG
protein levels were due to differences in transcriptional regulation
or transfection efficiency. Additionally, LNCaP cells both
transfected and not transfected with pDsRed empty vector showed
no SHBG immunoreactive band (Figure 6A).
Similarly, Western blot analysis of LNCaP transfected cells with
2 different pCDNA3.1 constructs (one including the exon 1A
59UTR sequence, SHBGEX1A-EX8-pCDNA3.1, and the other with
Figure 5C), showed that cells transfected with the construct
containing Ex2-Ex8 produced higher amounts of protein than
those transfected with Ex1A-Ex8 (Figure 6C, D). However,
Figure 1. Identification of uORFs in TU-1A and TU-1B. A) The upper panel contains a schematic representation of the SHBG gene. Coding
exons are shown in black boxes and noncoding alternative first exons are in gray boxes, with their respective sizes (in bp) indicated above. Below are
depicted the exon organization of mature mRNAs of TU-1A and 1B and the corresponding ORF organization. uAUGs (AUG1 and 2) are written in gray
while SHBG AUG is written in black (AUG3). uORFs are indicated in gray boxes while SHBG ORF is shown in a black box. AUG1 generates an uORF of 2
codons while AUG2 generates an uORF of 11 codons. B). Exon 2 scheme, showing AUG2 and 3. AUG2 starts a uORF whose stop codon overlaps the
SHBG translation start codon (AUG3).
SHBG Regulation by 59 UTRs
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analysis of SHBG Ex2-Ex8 mRNA levels by RT-PCR (25 cycles),
showed higher amounts of SHBG mRNA in cells transfected with
the construct containing Ex1A-Ex8 than in those transfected with
the Ex2-Ex8 construct (Figure 6C), indicating than the translation
efficiency of Ex1A-Ex8 mRNA was lower than that of Ex2-Ex8
(Figure 6C, D). Additionally, whereas no endogenous SHBG
mRNA was detected in non-transfected cells at the exponential
phase of the RT-PCR reaction (25 cycles) (Figure 6C), when the
number of PCR reaction cycles was extended to 35 (saturating the
reaction), the specific band was identified (Figure 6C), indicating
that the endogenous SHBG mRNA levels were much lower than
those in transfected cells, and therefore, protein levels would be
under the limit of detection of the western blot assay (Figure 6A,
C). As the molecular size of the SHBG immunoreactive bands was
identical in cells transfected with constructs with or without SHBG
59UTRs (Figure 6A, C), we concluded that the translation start site
Figure 2. Predicted secondary structure for 59UTR sequences of TU-1 and TU-1A. M-fold prediction of the 59UTR mRNA secondary
structure of TU-1 (A) and TU-1A (B). Predicted thermodynamic stability is shown, and the first SHBG codon is marked with a grey box. Junction
between exons 1A and 2 is indicated with a solid black line in B. The uAUG is shown in a blue box in B. The predicted major hairpin is also indicated
SHBG Regulation by 59 UTRs
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Figure 3. Predicted secondary structure for 59UTR sequences of TU-1B. M-fold prediction of the 59UTR mRNA secondary structure of TU-1B.
Predicted thermodynamic stability, the first SHBG codon, junction between exons 1B and 2, and the uAUG are indicated as in Figure 2. The presence
of hairpins is also indicated (HP1, HP2, HP3, HP4 and HP5).
SHBG Regulation by 59 UTRs
PLoS ONE | www.plosone.org5November 2010 | Volume 5 | Issue 11 | e13844
for exon 1A transcripts was localized in the previously suggested
first in-frame ATG of exon 2 (Met 30 of transcripts beginning with
the exon 1 sequence). To further support these results, 3
independent transient transfections of the SHBGEX1A-EX8-
pCDNA3.1 and SHBGEX2-EX8-pCDNA3.1 constructs were per-
formed in LNCaP and DU-145 prostate-derived cell lines
(Figure 7A, C) and SHBG protein levels were analyzed using
polyclonal AF2656 antibody. In both cell lines, SHBG protein
levels were again higher in cells transfected with the SHBG-
pCDNA construct excluding the exon 1A 59UTR sequence than
in those transfected with constructs including the SHBG 59UTR
(Figure 7A, B, C, D). Thus, the overall results supported that exons
1A and 1B were acting as 59UTRs that modulate SHBG protein
Application of the CART (classification and regression tree)
model established by Davuluri et al.  to SHBG 59UTR
sequences of exons 1, 1A and 1B enabled us to classify SHBG first
exons into 2 different classes (Table 1): exon 1 belonged to class III
(efficiently translated transcripts), whereas exons 1A and 1B
belonged to class I (poorly translated transcripts due to the
presence of stable secondary structures or uAUGs). None of the
SHBG 59UTRs analyzed corresponded to class II or TOP
(59terminal oligopyrimidines) mRNAs, whose translation is
regulated in a growth dependent manner .
The SHBG gene is composed of 13 different exons that
generate at least 6 different TUs, and a minimum of 19 different
transcripts [2,5]. Each TU is constituted by a common region
formed by exons 2 to 8, preceded by one alternative first exon.
Only one (exon 1) of the six alternative first exons described (exons
1, 1A, 1B, 1C, 1D, and 1E) presents an ATG in frame with the
SHBG coding sequence: TU-1 encodes a leucine-rich signal
peptide and is responsible for the production of plasma SHBG by
hepatocytes. Translation of TU-1A has been demonstrated in
human and mouse sperm containing the 11-kb human SHBG
transgene [6,8], and it has been suggested that TU-1A translation
starts at the first in-frame ATG of exon 2, which encodes
methionine 30 of transcripts beginning with exon 1 [7,8].
Translation of TU-1B, -1C, -1D, and -1E has not been previously
The presence of TU-1, -1A, -1B, -1C, -1D, and -1E has been
shown in human prostate [2,5]. We previously demonstrated that
TU-1B was the most abundant SHBG TU in the LNCaP, PC3,
and PZ-HPV7 cell lines , and that transcripts including exon 1
after exon 1A or 1B sequences were also found in prostate cell lines
and tissues, indicating that exon 1A/exon 1B and exon 1 can be
spliced together and are not always mutually exclusive . Herein,
we report the presence of SHBG mRNA corresponding to a
common region of all transcription units (Ex2-Ex5) in LNCaP,
PC3, and PZ-HPV7 cells, as well as mRNA corresponding to full-
length TU-1A and TU-1B in LNCaP cells and human prostate
tissue. With regard to their translation, although we were able to
identify the SHBG protein in human prostate, testis and plasma,
we could not detect its presence in LNCaP, PC3, and PZ-HPV7
cells or in their supernatant, except when SHBG was overex-
pressed in LNCaP cells with a Flag-tagged SHBG construct.
Moreover, when LNCaP cells were transfected with constructs
containing the putative non-coding exons 1A or 1B, the amount of
detected SHBG decreased considerably with respect to cells
transfected with constructs without these potentially noncoding
exons. Importantly, the molecular weight of the detected band did
not vary between the 2 groups, suggesting that exons 1A and 1B
were acting as 59UTR exons, regulating SHBG translation. These
results confirmed that the first in-frame ATG of the exon 2
sequence (which codes for methionine 30 of transcripts beginning
with the exon 1 sequence) acts as the first coding codon of TU-1A
and TU-1B. In this respect, it has been reported that regulation of
Table 1. SHBG classification of alternative 59UTRs.
#2 #234.5/.2.234.5 uAUGs
Exon 179 nt No
Exon 1A-2 158 ntNo
Figure 4. SHBG expression in human prostate cell lines. A) RT-PCR analysis of SHBG expression in human prostate-derived cell lines, using
primers that amplify a region common to all SHBG mRNA isoforms (Ex2-Ex5). B) RT-PCR analysis (Ex1A-Ex8) of exon 1A transcripts in LNCaP cells. The
different bands observed correspond to: a (full length transcript), b (skipping of exon 7) and c (skipping of exons 6 and 7). C) As previously reported
, full-length TU-1B transcripts are detected by RT-PCR analysis (Ex1B-Ex8) in human prostate tissue and LNCaP cells. Negative controls (Ctrl) are
performed with water instead of cDNA.
SHBG Regulation by 59 UTRs
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Figure 5. Western blot analysis of SHBG protein in human prostate. A–B) Two bands sized 52- and 48-kDa were detected in human prostate
tissue using the SHBG 11F11 antibody (A) and were identical in size to those detected in human plasma and human testis (B). C) SHBG constructs
used to transfect LNCaP cells. In the Flag-Ex2-Ex8 construct, SHBG ATG was mutated to ATA to avoid disturbing the Flag translation start site. The
nucleotides involved in the SHBG translation start site are shown in all the constructs. D2E) Western blot analysis of the SHBG protein using the
11F11 antibody in transfected and non-transfected human prostate-derived cell lines (D), and in the supernatant (E). In (E), plasma and the
hepatocarcinoma cell line (HepG2) were used as positive controls for secreted SHBG. F) Western blot analysis of LNCaP cells transfected or not with
Flag-SHBG and pDsRed-SHBG constructs.
SHBG Regulation by 59 UTRs
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SHBG Regulation by 59 UTRs
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translation initiation is a central control point in mammalian cells,
and that the rate of initiation limits translation of most mRNAs
. Translation regulatory elements in 59UTRs, such as uAUGs,
uORFs and complex mRNA secondary structures , are often
found in mRNAs encoding regulatory proteins like proto-
oncogenes, growth factors and their receptors, and homeodomain
proteins [14,21]. During embryonic development, the 59UTRs of
Antp, Ubx, RARb2, c-mos, and c-myc regulate protein levels in a
spatiotemporal manner, and translation initiation of several
growth factor mRNAs (IGFII, PDGF2, TGFb, FGF-2 and VEGF)
is specifically regulated during differentiation, growth, and stress
The presence of long 59UTRs containing uAUGs, uORFs, and
mRNA secondary structures reduces the efficiency of the scanning
process by impeding the ability of ribosomes to interact with the
59UTR in single-stranded form [18,21,22]. Studies in live cells
have determined that translation efficiency decreases abruptly
when hairpin stabilities in the 59UTR of genes increase from an
dG of 225 kcal/mol to 235 kcal/mol, and reach a basal
minimum as hairpins approach predicted thermal stabilities of
250 kcal/mol . In the 59UTR of TU-1A and TU-1B, we
identified 2 uORFs in TU-1A and 1 in TU-1B, as well as
thermodynamically stable secondary structures that might inter-
vene in the observed translation downregulation of these
transcripts. Furthermore, the greater decrease in translation of
TU-1B in comparison with TU-1A might be explained by a higher
composition of G/C nucleotides within the 59UTR sequence of
the former, its overall more stable secondary structure, and its
longer 59UTR sequence.
The regulatory role of exons 1A and 1B 59UTR on SHBG
translation has been further confirmed using the CART model for
classifying human 59UTR sequences . According to this
classification, the most relevant variables are the presence of
TOP (59 terminal oligopyrimidines), the secondary structure, UTR
length, and the existence of uAUGs . Use of the decision tree
multivariate analysis of CART for human 59UTR sequences
enabled us to classify transcripts presenting exon 1A-exon 2 and
exon 1B-exon 2 59UTR sequences as poorly translated transcripts
(Class I). This class mainly includes mRNAs encoding transcrip-
tion factors, growth factors, proto-oncogenes, and other regulatory
proteins that are poorly translated under normal conditions (eg, in
cells in resting state) . The inclusion of exons 1A and 1B
59UTRs in Class I correlated well with the transient transfection
results, in which addition of exon 1A or exon 1B 59UTR
sequences strongly downregulated translation from SHBG Ex2-
Ex8 constructs. Additionally, because Class I consists of genes
involved in regulation of cell growth and differentiation ,
inclusion of exon 1A 59UTR in Class I could also explain TU-1A
translation in germ cells of mice expressing human SHBG
transgene . On the other hand, SHBG TU-1 was classified as
efficiently translated transcripts (class III), which also correlated
well with easy detection of the secreted SHBG protein in human
plasma and in supernatant of HepG2 cell lines. None of the
59UTRs studied here were classified as class II 59UTRs which
correspond to TOP mRNAs, which are identified by a sequence of
6 to 12 pyrimidines at the 59end .
Selective stress-induced translational control involving uORF
has been demonstrated for GCN4, ATF4, ATF5, GADD34, and
PKCg . As SHBG exon 1A and 1B transcripts are poorly
translated under normal growth conditions, further experiments
should be performed to determine whether, under stress
conditions, translation of these transcripts is enhanced. Particu-
larly, in prostate cancer, it would be interesting to determine
whether the hypoxia environment and the increased oxidative
stress associated to tumor growth favor the translation of these
Another function of TU-1B might be the regulation of SAT2
gene expression, since it has been described that exon 1B overlaps
with the 59UTR sequence of the SAT2 gene , situated on the
negative strand of the chromosome 17, and therefore SHBG and
SAT2 genes would produce natural sense-antisense pair tran-
scripts that overlap head to head.
Regulation of SHBG translation through its 39UTR mRNA
sequence by miRNA cannot be ruled out. It has been estimated
that approximately half of the human genome is controlled by
miRNAs, since the human genome contains approximately 1000
miRNAs and each can control up to 10 mRNAs . A large
number of in vivo and in vitro studies have shown that miRNAs
either inhibit translation, destabilize mRNA, or both . Further
studies are required to investigate the contribution of miRNAs to
In conclusion, our results in human prostate-derived cell lines
indicate that SHBG TU-1A and TU-1B are translated from the
first in-frame ATG found in the exon 2 sequence, and that their
corresponding 59UTR exons downregulate SHBG translation.
All human cell lines were obtained from the American Type
Culture Collection (ATCC, Rockville, MD). Prostate cancer cell
lines LNCaP, PC3, and DU-145 were maintained in RPMI 1640
medium (PAA Laboratories, Pasching, Austria) containing 10%
fetal calf serum (PAA Laboratories) and supplemented with
penicillin/streptomycin, sodium pyruvate, and modified Eagle
media with nonessential amino acids, as recommended. The
hepatocarcinoma cell line HepG2 was maintained in DMEM
(PAA laboratories) containing 10% FCS and supplemented as
described above. Finally, the prostate cancer cell line PZ-HPV-7
was grown in keratinocyte-SFM medium (Invitrogen, Carlsbad,
CA), supplemented with 2.5 mg of EGF and 25 mg of bovine
pituitary extract (both from Invitrogen).
Human prostate tissue
Human prostate tissue was obtained from the non-tumoral part
of prostate carcinoma at the T2/T3N0M0 stage of patients
submitted to radical prostatectomy. Ethics approval for this study
was obtained from the Hospital Universitari Vall d’Hebron Ethics
Figure 6. SHBG translation is differentially modulated by alternative exons 1A and 1B. A) Western blot analysis of SHBG translation in
LNCaP cells transfected with different SHBG-pDsRed constructs, with and without different 59UTRs (exon 1A or exon 1B). Human plasma and Flag-
SHBG constructs were used as positive controls of SHBG protein detection, while LNCaP cells transfected with pDsRed empty vector and non-
transfected cells were used as negative controls. b-actin was used to normalize the quantity of protein loaded on the acrylamide gel. RT-PCR analysis
using primers that recognize a region common to all SHBG isoforms (Ex2-Ex8) was performed to normalize the transfection levels with the different
SHBG constructs. S18 primers were used to normalize cDNA levels loaded into the PCR reaction. B) Relative protein levels (RPL) and relative protein/
mRNA levels (RPML) are shown. RPL was calculated as a ratio of the b-actin level in the sample. RPML was obtained by dividing RPL by the quantified
intensity of mRNA SHBG bands. C) Western blot analysis of SHBG translation in LNCaP cells transfected with pcDNA 3-SHBG constructs with and
without the 59UTR (exon 1A). D) RPL and RPML were calculated as above.
SHBG Regulation by 59 UTRs
PLoS ONE | www.plosone.org9November 2010 | Volume 5 | Issue 11 | e13844
Figure 7. Transfection of pcDNA 3 constructs in LNCaP and DU-145 cell lines. A) Western blot analysis of SHBG translation in LNCaP cells
transfected with pcDNA 3-SHBG constructs with and without 59UTR (exon 1A). Three independent experiments were performed. B) RPL and RPML
were calculated. C) Western blot analysis of SHBG translation in DU-145 cells transfected with pcDNA 3-SHBG constructs with and without the 59UTR
(exon 1A). Three independent experiments were performed. D) RPL and RPML were calculated.
SHBG Regulation by 59 UTRs
PLoS ONE | www.plosone.org10November 2010 | Volume 5 | Issue 11 | e13844
Committee and informed written consent for participation in the
study was obtained in all cases, in keeping with the mentioned
Committee requirements. The histology of the prostate specimens
was evaluated by the urological pathologist.
To predict the secondary structure of SHBG 59 exons, we used
the MFOLD program (version 3.2) (http://mfold.bioinfo.rpi.edu/
cg1-bin/rna-form1.cgi)  and RNAfold webserver (http://rna.
tbi.univie.ac.at/cgi-bin/RNAfold.cgi) from the Vienna RNA
websuite . The optimal secondary structures for both
sequences were obtained in dot-bracket notation with minimum
Generation of SHBG Plasmid Constructs
Three types of constructs were generated: a) a Flag/SHBG
construct where the Flag tag is localized at the N-terminus end of
the fusion protein (FlagEx2-Ex8); b) two different tag less SHBG
pCDNA 3.1 constructs: one including the exon 1A 59UTR
sequence (Ex1A-Ex8-pCDNA3.1), and the other one without any
SHBG 59UTR sequence (Ex2-Ex8-pCDNA3.1); c) three different
SHBG/DsRed constructs where the DsRed tag is fused to the C-
terminus end of the SHBG protein: two including exon 1A or exon
1B SHBG 59UTR sequences (Ex1A-Ex8pDsRed or Ex1B-
Ex8pDsRed, respectively), and the third one without any SHBG
59UTR sequence (Ex2-Ex8pDsRed). While the Flag –SHBG
construct was used as a positive control of SHBG protein
translation as the Flag tag has its own translation start site, both
SHBG-pCDNA 3.1 and pDsRed constructs were used to test
SHBG protein translation by means of its own translation start
The different SHBG sequences were amplified by PCR from
cDNA of a human prostate sample using the primer pairs
described in the supplementary table (where the inserted
restriction sites are underlined). The PCR products were cloned
into the pRC 2.1 vector from the TOPO TA cloning kit
(Invitrogen, Carlsbad, CA) and then subcloned into the pCDNA
3.1 vector (kindly provided by Dr. Josep Roma), pDsRed 1N1
(kindly provided by Dr. Maurizio Scaltritti) or pCMV-Flag 6a
vectors (Sigma) using the corresponding restriction enzymes (New
England Biolabs, Ipswich, MA) (Table S1).
One day before transfection, LNCaP and DU 145 cells were
seeded in 100-mm culture dishes. The next day, cells were
transfected with 4 mg of plasmid DNA (pcDNA3.1-SHBG, SHBG-
pDsRed or Flag-SHBG) using Fugene 6 transfection reagent
(Roche, Basel, Switzerland), according to a standard protocol. The
medium was replaced with fresh medium 16 h post-transfection,
and protein and RNA extraction from transfected cells was
performed 48 h post-transfection. Transfected cells were exam-
ined for SHBG expression by RT-PCR and western blot.
RNA extraction and RT-PCR
Total RNA was isolated from human cell lines and tissues using
the RNeasy Mini/Midi Kit 50 (Qiagen). From each sample, 2 mg
of RNA were reverse transcribed using Superscript II H-
(Invitrogen), at 42uC for 50 min. One mL of the resulting cDNA
was amplified by PCR in non-saturating conditions using the
primers described in Table 2. Each PCR was performed in
triplicate. The PCR products were resolved by electrophoresis,
purified using the QIAquick gel extraction kit (Qiagen), cloned
using the TOPO TA cloning system (Invitrogen), and finally
sequenced using an ABI Prism 3100 genetic analyzer (Perkin-
Elmer Corp., Wellesly, MA).
Western Blot Analysis
Total protein from human cell lines and tissues was extracted
with RIPA buffer containing 150 mM Tris-HCl, 50 mM NaCl,
1% SDS, 1% Nonidet P-40, and 0.5% sodium deoxycholate,
complemented with a protease inhibitor cocktail (Sigma). Samples
were heat-denatured in loading buffer (Laemmli buffer and DTT)
and subjected to discontinuous SDS-PAGE with 4% and 10%
polyacrylamide in the stacking and resolving gels, respectively.
Proteins were transferred to Trans-Blot nitrocellulose membranes
(Biorad, Hercules, CA). Membranes were first blocked for 1 h in
TBS containing 0.01% Tween 20 (Sigma) and 5% skim milk, and
then incubated overnight at 4uC with primary antibodies against
human SHBG (SHBG mouse monoclonal 11F11 antibody, kindly
provided by Dr. Geoffrey Hammond; and SHBG goat polyclonal
AF2656 antibody, R&D Systems, Minneapolis, MN) in the same
buffer. Specific antibody-antigen complexes were identified using
horseradish peroxidase-labeled rabbit anti-mouse and goat anti-
rabbit IgG secondary antibodies (Dako, Glostrup, Denmark) and
then incubated with the chemiluminescent substrate West Dura
reagent (Pierce, Etten-Leur, Netherlands). The densitometry was
achieved using the Image J software.
To analyze SHBG secretion to the culture media, LNCaP, PC3,
PZ-HPV7, and HepG2 cells were seeded in 100-mm culture
dishes with the corresponding complete culture medium (see
above). After 24 h, the medium was replaced with fresh medium
without fetal bovine serum (FBS). Cells were incubated for 48 h
Table 2. List of primers used for RT-PCR.
Primer localizationPrimer sequence Annealing temperature/cycles
Exon 2 upper
Exon 5 lower
59 TGTCATGACCTTTGACCTCACC 39
59 TGAGATCTCGGCCTGTTTGTC 39
Exon 2 upper
Exon 8 lower
59 TGTCATGACCTTTGACCTCACC 39
59 AGGGGGGTTCTTAGGTGGAGC 39
Exon 1A upper
Exon 8 lower
59 TTCAAAGGCTCCCCCGCAGTGC 39
59 AGGGGGGTTCTTAGGTGGAGC 39
Exon 1B upper
Exon 8 lower
59 TGAAGAGCCTGAGAGAGCG 39
59 AGGGGGGTTCTTAGGTGGAGC 39
59 GATGGGCGGGGGAAAAT 39
59 CTTGTACTGGCGTGGATTCTGC 39
SHBG Regulation by 59 UTRs
PLoS ONE | www.plosone.org 11November 2010 | Volume 5 | Issue 11 | e13844
with FBS-free medium and 10-mL aliquots of each supernatant
were centrifuged in 30-mm6116-mm centrifugal concentrator
columns (Sartorius Stedim Biotech, Aubagne, France) at 30006g
for 30 minutes. Each concentrated medium was heat-denatured in
loading buffer and analyzed by western blot, as described above.
Twenty-five mg of total protein extract were N-deglycosylated
using N-glycosidase F enzyme (Roche). Reactions were performed
at 37uC O/N using 2 U of enzyme per 10 mg of protein in 0.25 M
of Tris-HCl (pH 7–9) in a final volume of 100 mL. Reaction
products were analyzed by western blot, as described above.
LNCaP cells were transfected with several different SHBG
contructs; in A and B) cells were transfected with constructs
containing the laminin-G-like N-or-C-terminal domains of the
SHBG protein tagged with hemagglutinin (HA) or green
fluorescent protein (GFP), respectively. Whereas anti-HA and
anti-GFP antibodies recognized both constructs, 11F11 antibody
only recognized the C-terminal domain. In (C), cells were
transfected with Flag-tagged constructs containing: a) the full
coding sequence, b) deletion of the last 22 amino acids (351 to
373), and c) deletion of exon 7. In this case, while anti-Flag
antibody recognized all the constructs, 11F11 antibody did not
recognize the exon 7-deleted construct. Similarly, in D) cells
transfected with the GFP-SHBG full-length sequence construct,
SHBG was detected by 11F11, whereas the GFP-SHBG construct
(exon 7 deleted) was not recognized by the antibody. Therefore,
the 11F11 antibody recognizes the C-terminal end of the SHBG
Identification of SHBG 11F11 antibody epitope.
protein, and specifically, the region coded by exon 7 and the
beginning of exon 8 (up to amino acid 351).
Found at: doi:10.1371/journal.pone.0013844.s001 (1.21 MB TIF)
terminal end of the protein. Parallelism between coding exons
(black boxes) and the amino acid sequence of SHBG monomer
(grey rectangle) is shown. The epitope recognized by 11F11
antibody is localized in the laminin-G-like C-terminal of SHBG
protein, specifically in the coded region between all of exon 7 and
the beginning of exon 8 (at least to amino acid 351 of mature
protein). The two N-glycosylation sites are indicated in black
boxes, while the O-glycosylation site is indicated with a black
Found at: doi:10.1371/journal.pone.0013844.s002 (0.26 MB TIF)
SHBG11F11 antibody epitope is localized at the C-
Found at: doi:10.1371/journal.pone.0013844.s003 (0.03 MB
List of primers used to generate SHBG plasmid
The authors would like to thank Marta Rebull (Unitat de Recerca
Biome `dica) for their technical support and Celine L. Cavallo for the
English revision of the manuscript.
Conceived and designed the experiments: TP ABD FM. Performed the
experiments: TP ABD. Analyzed the data: TP ABD AH ASM IdT JR FM.
Contributed reagents/materials/analysis tools: AH ASM IdT JR FM.
Wrote the paper: TP FM.
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