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J. Biol. Chem., Vol. 277, Issue 40, 37131-37138, October 4, 2002
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*
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From the European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
Received for publication, June 25, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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It is by now well established that the estrogen
receptor Although estrogens are primarily recognized as female sex hormones
that control the female secondary sexual characteristics, reproductive
cycle, and pregnancy they are also involved in the development and
maintenance of male reproductive organs (1) and in other physiological
processes such as liver, fat, and bone metabolism and in cardiovascular
and neuronal activity (2-4). The role of estrogens is also well
established in several pathological processes such as osteoporosis (5),
breast and endometrial cancers (6), and arteriosclerosis and
Alzheimer's disease (4). The effects of estrogens are mediated by
their intracellular receptors. To date, two estrogen receptors (members
of the nuclear hormone receptors superfamily) have been described:
estrogen receptor The estrogen receptor The alternative utilization of multiple promoters might serve to fine
tune ER In this study the effect of upstream AUGs (uAUGs) on the
translation of major ER Sequences Used in This Study--
GenBankTM
accession numbers of sequences used in this study are listed
below. Note that the mouse F 5'-UTRs result from splicing of the exon
F1 to the exon F2, respectively (10). The human sequences used
were: cDNA, NM_000125; A, NM_000125; C, X62462; E, X86816/AJ002561;
F, U68068/AJ002562; T, AJ421639. The mouse sequences used were:
cDNA, M38651; C, M38651; F1, AJ272164; F2, AJ272165.
Plasmid Construction and Site-directed Mutagenesis--
The
coding sequence of firefly luciferase was amplified from pGL3
basic plasmid (Promega) using the following primers: sense, 5'-GCGGGATCCATGACCATGGAAGACGCCAAAAACATAAAGAAAGGC-3';
antisense 5'-ATAGGGCCCTTACAATTTGGACTTTCCGCCCTTCTTGGCC-3'. The sense
primer introduced a BamHI site and initiator ATG sequence of
ER
The human ER
Site-directed mutagenesis was performed according to the procedure in
the QuikChange site-directed mutagenesis kit (Stratagene). All the
constructs were sequenced.
Cell Culture, Transfections, and Dual Luciferase
Assays--
MDA-231, 293, MCF-7, HepG-2, HeLa, and NIH-3T3 cell lines
were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), penicillin (100 units/ml, Invitrogen), streptomycin (100 µg/ml, Invitrogen), and
glutamine (2 mM, Invitrogen) at 37 °C in a 5%
CO2 incubator. The cells were split into 24-well plates
(for luciferase assays) or 6-cm dishes (for RNA isolation) such that
they were 50-80% confluent on the day of transfection. Transfections
were performed using Lipofectamine2000 transfection reagent
(Invitrogen) for NIH-3T3 cells or FuGENE 6 reagent (Roche Molecular
Biochemicals) for other cell lines following the recommendations of the
manufacturer. One hundred nanograms of pcDNA3.1-Luc vector
containing various 5'-UTRs and 100 ng of pSG5-Renilla vector were used
in each transfection. The reaction volumes were scaled up 5-fold for
6-cm dishes. In experiments evaluating the effect of ER
Twenty hours after transfection cells were lysed for 1 h with
Passive lysis buffer (Promega), and Dual-Luciferase assays (Promega) were performed following the recommendations of the manufacturer. Ten
microliters of cell lysate were transferred into a 96-well plate, and
the light emission was measured for 10 s with 2-s delay after the
injection of 50 µl of luciferase assay reagent or Stop&Glow reagent
on a EG&G Berthold Microplate Luminometer LB 96V. All experiments were
performed at least three times in triplicate.
RNA Isolation and RNase Protection Assays--
RNA from cells
transfected in the 6-cm dishes was isolated using the High Pure RNA
Isolation kit (Roche Molecular Biochemicals) according to the
manufacturer's procedure. After elution from the columns the RNA was
precipitated and redissolved to 1 mg/ml. RNase protection assays were
then performed as described previously (26). The RNase protection
probes for firefly luciferase and Renilla luciferase were
prepared as follows. A 133-bp fragment of firefly luciferase gene was
amplified from pGL3 basic vector (Promega) using the following sense
(5'-CGCGGATCCGGCGCCATTCTATCCTCTAG-3') and antisense
(5'-GCCGAATTCCCGCGTACGTGATGTTCACC-3') primers, which introduced
BamHI and EcoRI sites at the 5'- and 3'-end of
the amplified product, respectively. The PCR fragment was subsequently subcloned into pBlueScript II (SK Toeprinting Analysis--
The toeprinting analysis was performed
as described previously (27). Briefly, the constructs containing
various human and mouse ER Multiple Upstream ORFs in the Human and Mouse Estrogen Receptor
The human A 5'-UTR (the most GC-rich 5'-UTR, 72% GC) has the most
stable secondary structure, as predicted by Mfold version 3.12 (28), with a Gibbs free
energy Effect of the ER
Co-transfection with an ER Contribution of Individual uAUGs of Human T and Mouse F 5'-UTRs to
the Suppression of Downstream ORF Translation--
The mouse F and
human T 5'-UTRs, which reduced the translation from the downstream
luciferase ORF most efficiently, contain six and five uAUGs,
respectively. The contributions of individual uAUGs to the repression
of translation were evaluated using constructs with single uAUGs or
their combinations changed into UUG triplets (Fig.
3).
Interestingly, the mutation of the mouse F uAUG-1, -2, -3, or -5 did
not have a significant effect on the translation. It was only the
mutation of the uAUG-4 that was responsible for most of the 2.5-fold
increase in translation efficiency of the construct lacking all uAUGs.
Likewise, mutation of human T uAUG-1, -2, -3, -4, or -5 did not
increase the translatability of the 5'-UTR significantly. However, the
mutation of uAUG-6 improved the translation efficiency 6-fold. The
combined mutation of uAUG-5 and -6 increased the translation ~30-fold
and thus restored the efficiency of translation of this construct to
the level of the parental vector control. The additional elimination of
the uAUG-4, which is in the frame with the uAUG-5, did not further
improve the efficiency of translation. Nevertheless, the construct
lacking all uAUGs augmented the translation an additional 2-fold
above the level of the control.
Messages with Human or Mouse ER
The human T was further analyzed by stepwise mutation of the uAUG
codons into UUG codons starting at the 5'-end of the 5'-UTR and adding
the downstream uAUGs (Fig. 5). Such
additive removal of initiating codons caused ribosomes to initiate at
downstream uAUGs and resulted in a corresponding "shift" of the
toeprints further from the 5'-end of the mRNA. It is worth noticing
that the toeprints matching the main ORF AUGs were observed only after the removal of all uAUGs. The varying strength of the toeprints might
represent the potential of the particular AUG to serve as an initiating
codon. This mutational analysis also confirmed the specificity of the
observed toeprints. A toeprint not corresponding to any AUG was
observed in mutants in which more than the first three uAUGs
were removed (open arrow in Fig. 5). This might be caused by
a stabilization of the secondary structure that is normally disrupted
by the presence of the ribosome stalled at the first three uAUGs.
The ER Nevertheless, the possibility of cell-specific regulation of
translation efficiency needs to be considered (e.g. a uORF
in the retinoic acid receptor The toeprinting analysis showed that ribosomes initiate on multiple
upstream AUGs, and therefore the mRNAs containing various 5'-UTRs
of human or mouse ER If we use the same model as above, it might be expected that the human
E and F 5'-UTRs should not inhibit the translation of luciferase
significantly as their uAUGs are not in favorable Kozak context and all
uORFs end more than 100 nucleotides upstream of the main ORF (Fig. 1).
However, it was not anticipated that the human A 5'-UTR would not
inhibit the translation to an extent similar to mouse C or F 5'-UTRs as
the last uORFs of all three 5'-UTRs terminate in approximately the same
distance from the main ORF (54, 56, and 56 nucleotides, respectively).
A strong toeprint matching the A uAUG indicates that most ribosomes
initiate on this codon. Whether the secondary structure in human A
5'-UTR (72% GC) augments the recognition of the main AUG or another
mechanism such as internal ribosome entry is responsible for this
effect is not clear.
Finally, mutation of all uAUGs in human 5'-UTRs restored or even
augmented the translation of luciferase, whereas the mutants of mouse C
or F 5'-UTRs lacking all uAUGs restored the translation efficiency to
only 75 or 50% of the parental vector, respectively. It is possible
that a negative element present in mouse in the common part of the
5'-UTRs between the common acceptor splice site and main ORF, which is
only 70% identical between mouse and human, can negatively influence
the translation initiation from the main AUG. However, further research
is needed to clarify this issue.
In summary, we have shown in this study that multiple 5'-UTRs
positively and negatively influence the expression of ER
(ER) is transcribed from multiple promoters. One direct
consequence of multiple promoters is the generation of mRNA
variants with different 5'-untranslated regions (5'-UTRs).
However, the potential roles of these individual mRNA variants are
not known. All 5'-UTRs of ER contain between one and six upstream
open reading frames. In this study the effect of the 5'-UTRs of major
human and mouse ER mRNA variants on translation was evaluated.
Some of the 5'-UTRs were found to strongly inhibit translation of the
downstream open reading frame. Mutation of the upstream AUG codons
partially or completely restored translation efficiency. A toeprinting
analysis and assessment of the contribution of each AUG codon to the
inhibitory effect on translation showed that leaky scanning and
reinitiation occurs with these mRNAs. In conclusion, the upstream
open reading frames in the 5'-UTRs of ER mRNAs have the
potential to regulate estrogen receptor expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(NR3A1) (7) and estrogen receptor 
(NR3A2,
Nuclear Receptors Nomenclature Committee, 1999; Refs. 8 and 9).
(ER)1 gene is transcribed
from multiple promoters that give rise to mRNA variants with
distinct 5'-untranslated regions (Ref. 10 and references therein). In
total, seven (A, B, C, D, E, F, and T) and six (A, B, C, E, F, and H)
promoters are known for human and mouse ER gene, respectively, and
presumably these lists are not exhaustive. These promoters are utilized
in a tissue-specific manner resulting in different levels of expression of mRNA variants in individual tissues (11-16). The different
expression of human A, B, and C variants in normal and in cancerous
breast tissue and tumor-derived cell lines has also been observed (11, 17).
levels within the cell. This could occur at the level of
transcript production, transcript stability, or, as each variant has a
different 5'-UTR, efficiency of translation of the transcript. It is
known that in general the secondary structure and/or the presence of
AUGs and associated open reading frames (ORFs) in the 5'-UTRs can
regulate the translation of the main ORF (for review, see Refs.
18-20). There are two examples of such regulation in the nuclear
hormone receptor family. Several short upstream ORFs (uORFs) in the
mouse retinoic acid receptor 2 mRNA act to regulate the
tissue-specific expression of the receptor at the translational level
(21, 22). Recently the importance of a uORF for expression of the
glucocorticoid receptor has also been reported (23). The ER genomic
unit might be a good candidate for such regulation as it has a plethora
of mRNAs with different 5'-UTRs that contain between one and six uORFs.
mRNA variants in human and mouse was
analyzed in transient transfection assays and by in vitro
toeprinting. The 5'-UTRs of the human ER C, E, and F mRNAs
showed a moderate negative effect on the translation of a reporter
gene. However, human ER T and mouse ER C and F 5'-UTRs
significantly suppressed the translation of a reporter gene. Mutation
of the uAUGs partially or completely restored the translation
efficiency of these mRNAs. The extent to which different uAUGs
influence the translation of the downstream ORF with regard to their
homology to a consensus Kozak sequence (24) and the location of the
associated uORFs relative to the main ORF translation start site was
analyzed for the human T and mouse F variants. Toeprinting analysis
revealed that leaky scanning occurs with mRNAs bearing these
5'-UTRs and excluded the internal ribosomal entry site-mediated
translation as a major mechanism for translation of these mRNAs.
This study shows that 5'-UTRs might play an important role in the
regulation of ER expression in some target tissues.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(underlined) that when joined to luciferase forms a
BstXI restriction site. The antisense primer contained an
ApaI site at its 5'-end. The amplified product was
directionally cloned into the BamHI and ApaI
sites downstream of the CMV promoter in pcDNA3.1-Hygro(+) vector
(Invitrogen). The obtained construct, pcDNA3.1-Luc, was used as a
parental vector for subcloning various 5'-UTRs of ER. The 5'-UTRs of
human and mouse ER were amplified by reverse
transcription-PCR from human endometrium, liver, or testis and
mouse liver RNA using a common antisense primer for human
(5'-GGCGTCTTCCATGGTCATGGTAAGTGGCAGCCGGCG-3') and mouse
(5'-GGCGTCTTCCATGGTCATGGTAAGTGGCAGCCGGCG-3') that introduced the
beginning of the luciferase coding sequence behind the initiator ATG of
ER and formed the BstXI as described above. The 5'-UTRs of transcribed mRNAs contained 59 bp of vector sequence at their 5'-end followed by ER 5'-UTRs fused to luciferase coding sequence. The sense primers, containing an NheI site at their 5'-end,
were as follows: human A, 5'-CGCGCTAGCAGGAGCTGGCGGAGGGCG-3'; human C,
5'-CGCGCTAGCTTCACACACTGAGCCACTCGC-3'; human E,
5'-CGCGCTAGCAGTCAGAGAAATAATCGCAGAGCCTC-3'; human F,
5'-CGCGCTAGCACCAAAACTGAAAATGCAGGCTCCATGC-3'; human T, 5'-CGCGCTAGCCTCTTGCCTGCCGCCATGTAAGAAGC-3'; mouse C,
5'-CGCGCTAGCATCACACACCGCGCCACTCGATCATTCG-3'; and mouse F,
5'-CGCGCTAGCGAAAACACAAGGCTCCATGCTCAGC-3'. The resulting PCR
products were cloned into the NheI and BstXI
sites of pcDNA3.1-Luc.
expression vector plasmid HEO (pSG5-ER) was kindly
provided by P. Chambon (25). The mouse ER-expressing vector was
constructed by subcloning the ER coding sequence from pMT2-MOR
(kindly provided by M. G. Parker) into the EcoRI site of the pSG5 vector (25). The pSG5-Renilla vector was kindly provided by
M. Hentze and contains the Renilla luciferase coding sequence cloned between the SmaI and BamHI sites
of pSG5.
on
translation efficiency 100 ng of pSG5-ER vector was used.
) (Stratagene). Similarly, sense (5'-ACGGGATCCGGCCATGATTGGGGTGCTTG-3') and antisense
(5'-ATGGAATTCGGCCATTCATCCCATGATTCAATC-3') primers were used to amplify
a 120-bp fragment of Renilla luciferase from pRL-CMV plasmid
(Promega) that was subcloned into BamHI and EcoRI
sites of pBlueScript II (SK). Both constructs were linearized by
BamHI and transcribed with T7 RNA polymerase (Roche
Molecular Biochemicals) according to the manufacturer's instructions
in the presence of [-32P]dUTP (10 mCi/ml, 400 Ci/mmol,
Amersham Biosciences) using a ratio of hot to cold dUTP of 1:2.6. Dried
gels were exposed to BAS-MS phosphorimaging plates (Fuji) and scanned
on a Fuji FLA-2000 reader. The obtained signals were quantified using
Aida version 2.0 quantification software.
5'-UTRs upstream of the firefly
luciferase coding sequence (pcDNA3.1-Luc constructs) were
linearized by digestion with SfuI and transcribed using T7
RNA polymerase (Roche Molecular Biochemicals) according to the
manufacturer's instructions using 0.5 µg of linearized template in
the presence of 1 mM rATP, rCTP, and rUTP, 0.05 mM rGTP, and 1 mM CAP analog (New
England Biolabs). After a 10-min incubation at 37 °C rGTP was added
to a final concentration of 1 mM, and the reaction was
incubated further for 1 h. Transcribed RNA was extracted once with
phenol/chloroform, purified twice through Mini Quick Spin RNA columns
(Roche Molecular Biochemicals), precipitated by ammonium acetate, and
dissolved in water in a final concentration of 50 ng/µl. One hundred
nanograms of RNA were annealed with 20 pmol of primer
(5'-TTCACCTCGATATGTGCATCTGTAA-3') in 50 mM Tris, pH 7.5 by
heating to 65 °C for 2 min followed by a 5-min incubation at
37 °C. The annealed RNA/primer was added to the in vitro
translation reaction containing 33 µl of Flexi rabbit reticulocyte
lysate (Promega), 1 mM amino acids, 2 mM
dithiothreitol, 70 mM KCl, 40 units of RNasin (Promega),
and 3 mM cycloheximide or ethanol as vehicle.
No MgCl2 was added, and the internal concentration of
Mg2+ in the lysate was between 1.8 and 1.9 mM.
The reactions were incubated at 25 °C for 15 min and then placed on
ice. A 4-µl aliquot was analyzed by primer extension in a 20-µl
reaction volume containing 50 mM Tris, pH 7.5, 40 mM KCl, 6 mM MgCl2, 10 mM dithiothreitol, 0.625 mM dNTPs, 20 units of
RNasin, 2.5 mM cycloheximide, and 100 units of Superscript
(Invitrogen). The reactions were incubated for 45 min at 30 °C, the
reaction was stopped by extraction with 20 µl of phenol/chloroform,
and 5 µl of the upper phase were separated on a 6% denaturing
polyacrylamide gel (7 M urea). For sequencing reactions, the plasmids were annealed with the same primer and sequenced using the T7 Sequenase kit (Amersham Biosciences) according to the manufacturer's instructions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5'-UTRs--
In the following text, ER mRNA variants are
designated by their capital letter, and the uAUGs and uORFs are
designated by their distal position from the 5'-end of the mRNA.
The 5'-UTRs of major human and mouse ER mRNA variants used in
this study are illustrated in Fig. 1. The
lengths of the 5'-UTR variants are between 189 (human C) and 327 nucleotides (human F). Sequence analysis revealed the presence of AUGs
and associated ORFs upstream of the main ORF translation start site in
all variants. The numbers of uAUGs are: one in human A and E and mouse
C, two in human C, four in human F, five in mouse F, and six in human
T. The mouse F uAUG-4 has the most favorable Kozak context for
translation initiation ((A/G)CCAUGG, Ref. 24) having an A at position
3 and G at position +4. The other uAUGs are in a similar or a less favorable translation initiation context compared with the two initiation AUGs of the main ORF. The length of the associated uORFs
varies from a single codon (human E, F4, and T1) to 111 codons (human
T4). The human C2, T4, T5, and T6 uORFs extend beyond the initiation
AUG into the ER ORF.
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Fig. 1.
Schematic representation of the human and
mouse estrogen receptor 5'-UTRs used in this
study. On the top the genomic organization of the
5'-region of the ER gene is depicted as an upstream exon that is
spliced to the common acceptor splice site located in the first coding
exon of ER (exon 1). The exon 1 of ER contains the
translational start site (ATG) and also encodes the untranslated region
(dark gray). All mRNA variants share the untranslated
region between the common acceptor splice site and the main ATG.
Promoters are shown as broken arrows. Numbers
below show the location of major features in human and mouse
according to Refs. 7 and 41, respectively. The 5'-region of human and
mouse ER mRNA variants used in this study is shown
below. The shared part of the untranslated region is
depicted in dark gray (see above), and the part encoded by
upstream exons is depicted in light gray. The sequence
context of individual AUGs is shown above the schemes.
Numbers below the schemes show the position of major
features in each individual mRNA variant. Arrows
represent the upstream ORFs with their terminator position at the
arrowhead. cds, coding sequence of
ER.
G = 114.98 kcal/mol and the most stable
individual hairpin with a G of only 17.7 kcal/mol. The
most stable hairpin was identified in the human F 5'-UTR with G = 20.6 kcal/mol. Secondary structures with free
energy higher than G = 30 kcal/mol are not
considered to impair translation (29-32). Therefore it is likely that
none of the 5'-UTRs substantially inhibits translation of the
downstream ORF due to their secondary structure.
5'-UTRs on Translation of Downstream Open
Reading Frame--
To evaluate the effect of various 5'-UTRs on
translation of the main ORF, the constructs containing different
5'-UTRs placed upstream of a luciferase reporter gene were transiently
co-transfected with a Renilla luciferase-expressing plasmid
into ER-negative human breast carcinoma cell line MDA-231MB (human
5'-UTRs) or mouse fibroblast cell line NIH-3T3 (mouse 5'-UTRs) (Fig.
2). The parental vector has a 90-bp
5'-UTR devoid of any uAUGs and thus served as a control for translation
efficiency. The firefly luciferase values were corrected for
transfection efficiency using the Renilla luciferase
activity and also with mRNA expression in transfected cells (RNase
protection assays). The human A 5'-UTR increased the translation of
luciferase ~1.3-fold compared with the parental vector. The human C,
E, and F 5'-UTRs had a rather moderate negative effect on translation
efficiency. However, the human T and mouse C and F efficiently reduced
the translation of the luciferase to 3, 40, or 20% of the wild type,
respectively. Mutation of all upstream AUGs to UUGs restored or even
enhanced the translation efficiency of all human 5'-UTRs but not those
of mouse. The removal of all uAUGs had an especially pronounced effect
on the human T where it increased the translation by more than 60-fold.
Similar results were obtained using other cell lines such as HeLa,
MCF-7, HepG2, and 293 (data not shown).
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Fig. 2.
The effect of the human and mouse
ER 5'-UTRs on translation. The relative
luciferase activity of cells transfected with constructs containing
different human and mouse ER 5'-UTRs and their mutated versions. The
activity of the parental vector was set to 100%. Error bars
show the standard deviation of three independent experiments
performed in triplicates. The RNase protection assays used to quantify
the amounts of mRNAs expressed in transfected cells are shown
below the graph. wt, wild type.
-expressing vector or transfection into
the ER-positive cell line MCF-7 was performed to determine whether
ER itself has an effect on translation of different mRNA variants. However, no change in translation efficiency has been observed either in the presence or in the absence of estradiol (data
not shown). These results show that upstream AUG triplets in 5'-UTRs of
the multiple mRNA of ER variants can regulate the expression on
the level of translation.
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Fig. 3.
The contribution of individual uAUGs of the
mouse F and human T ER 5'-UTRs to translation
repression. The relative luciferase activity of cells transfected
with constructs lacking a single or combination of uAUGs of the mouse F
(left) and of the human T (right) 5'-UTRs. The
activity of the parental vector was set to 100%. Error bars
show standard deviation of three independent experiments performed in
triplicates. The schemes of the constructs used are shown
above the graphs. The RNase protection assays used to
quantify the amounts of mRNAs expressed in transfected cells are
shown below the graphs. mER, mouse ER;
hER, human ER; wt, wild type;
mut, mutated.
5'-UTRs Are Translated by Leaky
Scanning--
To determine the initiation AUG codons in mRNAs
containing various 5'-UTRs of human and mouse ER the ribosome
toeprinting analysis was performed (Fig.
4). Multiple toeprints matching one to
four uAUGs were observed in all cases indicating that these mRNAs
are translated by leaky scanning. A fraction of ribosomes passed the
first AUGs, presumably due to a weak Kozak context, and initiated on
the subsequent AUG codons. In the case of human A and E weak toeprints
corresponding to the main ORF AUGs were also observed. Several
toeprints that were induced by addition of cycloheximide but that do
not correspond to any AUG (or GUG) were also observed (indicated
by white asterisks in Fig. 4). Their origin is unclear.
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Fig. 4.
Toeprinting analysis of initiating AUGs in
human and mouse ER mRNAs. Black
asterisks indicate toeprints induced by addition of cycloheximide,
and their corresponding AUG codons are listed on the right.
White asterisks indicate toeprints that do not match any AUG
codons. A molecular weight marker is shown on the left.
CHX, cycloheximide.
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Fig. 5.
Toeprinting analysis of initiating AUGs in
human T ER mRNA. The hER T 5'-UTR
is depicted on the right with arrows representing
short upstream open reading frames. The position of a primer used for
extension is shown. The upstream AUGs are listed on the
right with large black arrows pointing to the
corresponding toeprints. Open arrows represent toeprints of
unknown origin. On top, numbers of the AUG codons
mutated to UUG are shown. wt, wild type; ctrl,
control; CHX, cycloheximide; cds, coding sequence
of the firefly luciferase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mRNAs are transcribed from multiple promoters that
are distributed over more than 150 kilobase pairs of genomic region (10). We have shown that the expression of the ER in human and mouse
is regulated by 5'-UTRs on the level of translation. Whereas the human
A, C, E, and F 5'-UTRs do not substantially inhibit translation of
downstream ORF, the mouse C and F and especially the human T 5'-UTRs
exhibit inhibitory effects to various extents (13-16). This finding
contributes to a more complete understanding of the mechanisms by which
the desired levels of the ER protein are achieved in target cells
given the observed tissue-specific expression of ER mRNA
variants (11). For example, the major mRNA variants expressed in
human mammary gland and endometrium (well known estrogen target
tissues) are A and C (13). The results presented here show that human C
5'-UTR has very little inhibitory effect, while human A even augments
the translation of the main ORF. Thus A and C ER promoters might be
specifically utilized in these tissues in order to provide relatively
high levels of ER protein. On the contrary, the human T 5'-UTR,
which is specifically expressed at low levels in testis (33),
suppresses translation very efficiently. It is known that estrogen
receptors are important for proper testis development and for normal
spermatogenesis as exposure to excessive levels of estrogens or
phytoestrogens during fetal and neonatal development leads to male
reproductive disturbances (see Ref. 34 and references therein). Tight
regulation of low ER expression in testis is likely to be necessary.
The weak translational efficiency of the human T mRNA might ensure
that low levels of ER are expressed in the target cell.
2 mRNA represses its translation
in heart and brain but not in other tissues; Refs. 21 and 22). The
extent to which 5'-UTRs influence the translation might also vary
during certain stages of development or may depend on external signals.
The experiments performed here in a small range of different cell types
might not be adequate to detect such variations. It has also been
reported in the literature that peptides encoded by short uORFs can
regulate expression of the retinoic acid receptor 2 (see above) or
the glucocorticoid receptor (22, 23). However, none of the ER
uORFs is homologous to any of these peptides. Furthermore, although the
number and position of most uORFs in different 5'-UTRs is relatively
well conserved between mouse and human, most of these share no
homology. Only human C2 and mouse C are partially homologous; however,
mouse C is significantly shorter (36 and 18 amino acids, respectively).
Therefore, it seems unlikely that peptides encoded by uORFs in various
ER 5'-UTRs would influence the expression of ER.
are translated by a leaky scanning mechanism.
Furthermore, the data obtained with mouse F and human T 5'-UTRs support
the scanning and reinitiation model of translation (35) of these
mRNAs (36). In this model the 40 S ribosomal subunits
attaches to the cap and scans along the mRNA until it reaches the
first AUG codon where it initializes translation. If the first AUG is
not recognized (e.g. due to weak Kozak context or secondary
structure) the ribosome may pass this AUG and initiate at another AUG
downstream, resulting in leaky scanning (for review, see Refs. 20 and
37). After translating the ORF, the ribosome can reinitiate at another
downstream AUG, detach from the mRNA, or stall at the termination
codon of the ORF. The efficiency of reinitiation on a downstream AUG
depends also on the distance between the end of upstream ORF and the
AUG (38-40). When this distance is short (less than 79 nucleotides;
Ref. 38) the ribosome is less likely to recruit the necessary
initiation factors before it reaches the next AUG, and consequently it
fails to initiate on the downstream AUG. This model accounts for the
contribution of individual uAUGs of the human T 5'-UTR to inhibition of
translation efficiency (Fig. 3). This 5'-UTR contains six uORFs (Fig.
1). The first three uORFs end far upstream of the main ORF, and the scanning/reinitiation model would predict that they have only a
moderate effect on translation efficiency. This is confirmed by
mutation of individual uAUG-1, -2, or -3, which did not dramatically improve the translation of luciferase (~1.5-fold). On the contrary, translation of uORF-4 or -5 (which are in-frame to each other) or
uORF-6 would be predicted to strongly inhibit translation of the main
ORF as they all terminate downstream of the main ORF initiator AUG.
Moreover, the translation of the uORF-2 would affect initiation on
uAUG-4 allowing the ribosome to pass and reach uAUG-5 or -6. The
mutation of uAUG-4 or -5 would have a minimal effect in the presence of
uAUG-6. The results confirm this prediction. The mutation of uAUG-6
improved translation of luciferase ~6-fold, and combined mutation of
uAUG-5 and -6 improved translation more than 30-fold. The additional
mutation of uAUG-4 did not have any effect. The additional mutation of
the first three uAUGs (i.e. construct lacking all uAUGs)
further increased the translation only 2-fold, reaching a
60-fold increase compared with the wild type sequence. Similar to the
human T 5'-UTR, the mouse F 5'-UTR contains a large number of uORFs.
The uORF-1 and uORF-2 terminate far upstream of the main ORF. The
uORF-3 translation would be expected to be inhibited by the uORF-1 and
uORF-2. The uAUG-4 has a favorable Kozak context. Consequently, the
uORF-4 would affect the translation of the main ORF the most
efficiently. The mutation analysis again confirmed this prediction
(Fig. 3). Moreover, the toeprinting analysis showed that the
translation initiation indeed occurs at the first several uAUGs
in all 5'-UTRs tested. Therefore, the ribosomes reinitiate downstream
at either another uAUG or the AUG of the main ORF.
in human
and mouse. The findings that human A and C are translated with a high
efficiency while human T strongly inhibits the translation of the
downstream ORF are especially physiologically relevant. Most of the
observed effects are caused by upstream ORFs. Furthermore, we have
shown that mRNAs containing human or mouse ER 5'-UTRs are
translated by leaky scanning and reinitiation.
| |
FOOTNOTES |
|---|
* This work was supported in part by European Community Grant QLK6-1999-02108.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: European Molecular
Biology Organization, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Tel.: 49-6221-8891102; Fax: 49-6221-8891200; E-mail: Frank.
Gannon{at}embo.org.
Published, JBC Papers in Press, July 29, 2002, DOI 10.1074/jbc.M206325200
2 Mfold server: bioinfo.math.rpi.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
ER, estrogen
receptor ;
UTR, untranslated region;
ORF, open reading frame;
uORF, upstream ORF;
uAUG, upstream AUG;
CMV, cytomegalovirus.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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