From the Programa de Biomedicina Molecular, Escuela
Nacional de Medicina y Homeopatía del Instituto
Politécnico Nacional and the Departments of
§ Biomedicina Molecular and ¶ Patología
Experimental, Centro de Investigación y de Estudios Avanzados del
Instituto Politécnico Nacional,
CP 07300, México, Distrito Federal
Received for publication, November 18, 2002, and in revised form, January 27, 2003
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ABSTRACT |
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The multidrug resistance (MDR) phenotype in
Entamoeba histolytica is characterized by the
overexpression of the EhPgp5 gene in trophozoites grown in
high drug concentrations. Here we evaluated the role of
EhPgp5 mRNA stability on MDR using actinomycin D. EhPgp5 mRNA from trophozoites growing without emetine
had a half-life of 2.1 h, which augmented to 3.1 h in cells
cultured with 90 µM and to 7.8 h with 225 µM emetine. Polyadenylation sites were detected at 118-, 156-, and 189-nucleotide (nt) positions of the EhPgp5 mRNA 3'-untranslated region. Interestingly, trophozoites grown with
225 µM emetine exhibited an extra polyadenylation site at 19 nt. The 3'-untranslated region sequence is AU-rich and has putative
consensus sequences for RNA-binding proteins. We detected a RNA-protein
complex in a region that contains a polypyrimidine tract (142-159 nt)
and a cytoplasmic polyadenylation element (146-154 nt). A longer
poly(A) tail in the EhPgp5 mRNA was seen in
trophozoites grown with 225 µM emetine. Emetine stress
may affect factors involved in mRNA turnover, including
polyadenylation/deadenylation proteins, which could induce changes in
the EhPgp5 mRNA half-life and poly(A) tail length.
Novel evidence on mechanisms participating in E. histolytica MDR phenotype is provided.
Entamoeba histolytica, the protozoan parasite
responsible for human amoebiasis, presents the multidrug resistance
(MDR)1 phenotype (1)
described first in mammalian cells (2) and then in several protozoan
parasites (3, 4). MDR is associated with the overexpression of a
170-kDa membrane molecule known as P-glycoprotein (PGP), an
energy-dependent pump that extrudes drugs from the cells
(5, 6). In E. histolytica, MDR phenotype is given mainly by
overexpression of the EhPgp1 and EhPgp5 genes, which are finely regulated by transcriptional factors (7-9). Although
EhPgp1 is constitutively expressed in drug-resistant trophozoites of clone C2, EhPgp5 gene is overexpressed only
when C2 cells are grown in a high emetine concentration (10, 11). Both
genes are also amplified in the presence of a high drug concentration (12).
Transcriptional regulation of eukaryotic mdr genes has been
considered as the major control point for PGP synthesis, although gene
amplification mechanisms also participate in this event (12, 13).
Moreover, there is growing evidence of pivotal post-transcriptional (14-17) and post-translational (18-20) regulation of the PGP
expression. On the other hand, mRNA stability has recently emerged
as a critical control step in determining cellular stationary mRNA
levels. The abundance of a particular mRNA can fluctuate many folds
due to alterations in mRNA stability without any change in the
transcription rate (21). The mRNA half-life is determined by a
complex set of protein interactions at the 3'-untranslated region
(3'-UTR) depending on conserved cis-element sequences and secondary
structures (for review, see Ref. 22). The 3'-UTR also contains
consensus sequence elements that mediate mRNA nuclear export,
cytoplasmic localization, translation efficacy, and polyadenylation
control (23, 24). The pre-mRNAs are polyadenylated in a reaction
involving 3' endonucleolytic cleavage followed by poly(A) tail
synthesis (25). Poly(A) tail is also a modulator of mRNA stability
and translation (26, 27). Strict control of poly(A) tail length is
achieved by the concerted interplay of key factors, including poly(A)
polymerase, deadenylases, and poly(A)-binding protein activities
(25).
Several reports have addressed the importance of mRNA
stability on the mdr genes expression regulation.
Pgp1, Pgp2, and Pgp3 mRNAs have a
higher half-life in rat tumor cells than in normal cells (15), whereas
rat MDR hepatocytes in culture present a higher amount of PGP2 protein
due to a post-transcriptional mechanism controlling mRNA stability
(14). Human MDR1 mRNA has a half-life of 30 min, which
is prolonged to more than 20 h upon treatment with cycloheximide,
suggesting that protein synthesis inhibition may influence the
stability of certain mRNAs (16, 17). However, molecular mechanisms
controlling mdr mRNA stability remains to be elucidated.
In E. histolytica, mRNA stability mechanisms have not
been studied yet. The presence of higher levels of EhPGP5 protein in the multidrug-resistant trophozoites of clone C2 could be influenced by
both transcriptional activation and increased mRNA stability. In
this paper, we measured the EhPgp5 mRNA half-life in
trophozoites of clone C2 grown at different emetine concentrations. Our
data showed that EhPgp5 mRNA stability is increased at
high emetine concentrations, indicating that mRNA half-life is also
regulating the MDR phenotype. In addition, here we initiated the study
of the mechanisms involved in mRNA turnover in this parasite.
E. histolytica Cultures--
Trophozoites of the clones A
(drug-sensitive) and C2 (drug-resistant) (strain HM1:IMSS) (28) were
axenically cultured in TYI-S-33 medium (29). Trophozoites of clone C2
were cultured without emetine (C2) or with 90 (C2(90)) and 225 (C2(225)) µM emetine. Logarithmic phase growing cultures
were used in all experiments. All assays presented here were performed
at least three times by duplicate.
Transcriptional Inhibition by Actinomycin D--
Actinomycin D
(Roche Molecular Biochemicals) dissolved in dimethyl sulfoxide
(Me2SO) (0.5 mg/ml) was added to the trophozoites cultures
to a final concentration of 10 µg/ml of medium, and cells were
incubated at 37 °C for different times. Fresh medium supplemented with [3H]UTP (10 µCi/ml) was added to the actinomycin
D-treated trophozoites for 2 more hours in the absence of
actinomycin D. Immediately, total RNA was isolated by TRIzolTM
(Invitrogen). Incorporation of [3H]UTP in 20 µg of
total RNA was measured by liquid scintillation counting system
(Beckman) in duplicate samples, and data obtained were plotted.
Cytotoxicity of actinomycin D and Me2SO was checked by cell
viability using trypan blue and measuring the growth rate of the
treated cultures.
Reverse Transcriptase (RT)-PCR Experiments--
100 ng of total
RNA from trophozoites of clones A, C2, C2(90), and C2(225) were
preincubated at 37 °C for 15 min with 10 units of RNase-free DNase I
(Stratagene). Single-stranded cDNAs were synthesized using 10 mM each dNTP and 100 ng of oligo(dT18) in diethyl pyrocarbonate-treated water. The mixture was heat-denatured at
65 °C for 5 min and quick-chilled on ice. Then we added buffer used
to synthesize the first-strand (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2), 100 mM dithiothreitol, 200 units of SuperscriptTM
II RNase H EhPgp5 mRNA Stability Assays--
Total RNA from
trophozoites of clones C2, C2(90), and C2(225) was obtained at 0, 2, 4, 8, and 12 h after actinomycin D-induced transcriptional blockage. EhPgp5 and actin mRNAs were
measured by multiplex RT-PCR as described above, and intensity of the
bands in ethidium bromide-stained gels was quantified by densitometric analysis in a PhosphorImager apparatus (Personal Molecular Imager FX,
Bio-Rad). The pixels given by the actin transcript in trophozoites of
clones C2, C2(90), and C2(225) without treatment
(t0) were taken as 100% in each clone.
EhPgp5 mRNA levels were normalized with respect to the
actin amount in each lane. Experimental EhPgp5 mRNA
half-life (the time at which 50% of mRNA molecules remained intact) was determined by plotting the EhPgp5 mRNA
amount at different times on a semilogarithmic scale. In these
estimations the EhPgp5 mRNA amount at
t0 was taken as 100% in each clone. Theoretical half-life of the EhPgp5 mRNA was obtained from the
logarithmically transformed best-fit line by linear regression analysis
using the decay equation t1/2 = ln 2/K, where K corresponds to the decay constant (32).
S1 Nuclease Mapping Experiments--
20 µg of total RNA from
trophozoites of clones C2, C2(90), and C2(225) were hybridized with a
697 bp of [ In Vitro Transcription--
Templates for transcript synthesis
were prepared from pBluescript II SK (+/ Cytoplasmic Extracts--
Cytoplasmic extracts (CE) were
obtained as described (33) with some modifications for E. histolytica. Briefly, 1 × 106 trophozoites of
clones C2, C2(90), and C2(225) were washed with 1 ml of
phosphate-buffered saline, pH 6.8, supplemented with 10 mM
HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM
KCl, 0.5 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride and incubated on ice for 15 min. Then 25 µl of 10% Nonidet P-40 and protease inhibitor mixture (0.5 mM phenylmethylsulfonyl fluoride, 2 mM
benzamidine, 5 µg/ml each aprotinin, pepstatin A, leupeptin, and
E-64) were added, and samples were vortexed for 10 s and
centrifuged at 12,000 × g at 4 °C for 1 min. The
supernatant was mixed with 0.11 volume of 100 mM HEPES, pH
7.9, 30 mM MgCl2, 250 mM KCl
solution and centrifuged at 14,000 × g at 4 °C for
1 h. Protein concentration was determined by the Bradford
method (34).
RNA Electrophoretic Mobility Shift Assays--
5 × 105 cpm of each PSI19, PSII118,
PSIII156, and PSIV189
[ Analysis of Poly(A) Tail Length--
Total RNA was used for
ligase-mediated poly(A) test (LM-PAT) according to the method described
(35). 1 µg of total RNA was incubated at 37 °C for 15 min with 10 units of RNase-free DNase I (Stratagene). Annealing and in
situ ligation of 20 ng of oligo(dT18) to the 3' end of
poly(A) tails of total RNA were done at 42 °C for 30 min in diethyl
pyrocarbonate-treated water. Subsequent annealing of 200 ng of hybrid
oligo(dT18)-adapter primer
(5'-GACTCGAGTCGACATCGAT18-3') was done at 12 °C for
2 h. cDNA synthesis was performed using the first-strand
buffer, 100 mM dithiothreitol, 200 units of
SuperscriptTM II, and recombinant RNasin ribonuclease
inhibitor (40 units) at 42 °C for 1 h. PCR was performed using
1/10 volume of the samples and the (5'-CTGAGCTCAGCTGTAGCT-3')
antisense primer complementary to the adapter region of the
oligo(dT18)-adapter primer and the EhPgp5-3'-UTR-S sense oligonucleotide. An actin 3'-UTR
(5'-GATCAATTCTTGCCTCAT-3') sense primer was used as a control.
Amplification for Ehgp5 and actin genes was done in 30 cycles at 95 °C at 30 s, 59 °C at 35 s, 72 °C at
90 s, and a final extension step at 72 °C for 7 min. PCR
samples were separated on a 1.5% Tris-buffered EDTA-agarose gel then
transferred to nylon membranes and hybridized with specific EhPgp5 and actin 3'-UTR [ Actinomycin D Inhibits E. histolytica Transcription--
To study
the mechanisms controlling mRNA decay in E. histolytica
we first investigated the effect of the transcription inhibitor actinomycin D on viability, growth, and mRNA synthesis in
trophozoites of the drug-sensitive clone A and drug-resistant clone C2.
Cell viability of trophozoites of clone C2 incubated 12 h with or
without actinomycin D was 98%. At this time cell growth was slightly
delayed in the actinomycin D-treated trophozoites in
comparison with untreated cells (Fig.
1A). The effect of actinomycin
D on cell growth and viability was similar in trophozoites of all
clones tested (data not shown). Then we performed experiments to
determine whether mRNA synthesis was affected by actinomycin D and
the time required to inhibit at least 90% of mRNA synthesis.
Results showed that actinomycin D affects the [3H]UTP
incorporation into new synthesized RNA in a time-dependent manner (Fig. 1B). Untreated trophozoites of clones A and C2
incubated for 2 h (t0) with
[3H]UTP incorporated 16,450 and 14,660 cpm, respectively.
Each value was taken as 100% incorporation for the corresponding clone
(Fig. 1B). Trophozoites of clones A and C2 preincubated for
30 min with the drug and then incubated with [3H]UTP for
2 h incorporated 50.4 and 59.9% of radioactivity, respectively. One hour later, both clones presented only 12% incorporation of [3H]UTP (Fig. 1B). These low levels of RNA
synthesis were maintained in trophozoites of both clones incubated with
actinomycin D for up to 8 h (Fig. 1B). As a control for
mRNA integrity, equivalent amounts of total RNA were isolated at
each time, and rRNA were visualized in ethidium bromide-stained agarose
gels (Fig. 1C). No appreciable changes in the rRNA amount
were observed, indicating no significant RNA degradation. These data
provided us a temporal window in which the mRNA decay could be
analyzed.
EhPgp5 mRNA Stability Is Higher in Trophozoites of the Clone C2
Cultured with Emetine--
To investigate the EhPgp5
mRNA stability in trophozoites growing with different emetine
concentrations, we determined the EhPgp5 mRNA half-life
in actinomycin D-treated trophozoites of clones C2, C2(90),
and C2(225). EhPgp5 mRNA was measured from 0 to 12 h by semiquantitative RT-PCR assays in total RNA. We included actin
primers in all reactions as internal control. Results showed that
EhPgp5 mRNA was present in untreated trophozoites of
clone C2, and the signal diminished progressively at 2 and 4 h
after the transcriptional blockage (Fig.
2A). In clone C2(90), the
EhPgp5 transcript was detected up to 8 h after
actinomycin D treatment (Fig. 2B). Interestingly, in clone
C2(225) the EhPgp5 mRNA was detected even 12 h
after the transcriptional blockage (Fig. 2C). These data
indicated that EhPgp5 mRNA amounts are reduced in a time-dependent manner in actinomycin D-treated
trophozoites, but they are maintained for a longer time in the
emetine-cultured cells. In contrast, we did not detect the
EhPgp5 mRNA in untreated trophozoites of the wild type
drug-sensitive clone A (Fig. 2G), confirming that
EhPgp5 gene is not transcribed in the drug-sensitive trophozoites (36).
The EhPgp5 and actin mRNAs were quantified by
densitometry and arbitrary expressed in pixels (Fig. 2,
D-F). Pixels given by actin at t0
were taken as 100% in each clone, and the EhPgp5 percentage was expressed with respect to the actin mRNA levels. At
t0, the actin amount appeared almost unaltered,
whereas EhPgp5 varied in the three clones. In trophozoites
of clone C2, the EhPgp5 mRNA was 63% of the actin
mRNA (Fig. 2D), and in C2(90) it was 81% (Fig.
2E), whereas clone C2(225) exhibited similar levels of both transcripts (Fig. 2F). The other bands seen in the gels were
not related to the EhPgp5 transcript, as probed by Southern
blot hybridization using the EhPgp5 probe (data not shown).
To determine the experimental mRNA half-life, the results of
normalized EhPgp5 mRNA levels were plotted in a
semilogarithmic scale against the exposure time to actinomycin D (Fig.
2H). In these calculations, the amount of EhPgp5
and actin mRNA at t0 was taken as 100% in
each clone. In trophozoites of clone C2, the experimental
EhPgp5 mRNA half-life was estimated in 2.1 h, whereas in C2(90) it was 3.1 h and 7.8 h in C2(225),
confirming significant variations in the decay rates of the three
clones. In addition, these experiments showed that actin mRNA decay
remained with minimal changes during the 12-h course of the
transcription inhibition in all clones. There are reports proposing
that actin mRNA has a half-life between 24 and 33 h in
mammalian cells (37). According to our experiments, E. histolytica actin mRNA has a half-life longer than 12 h.
Experimental values were close to the theoretical EhPgp5
mRNA half-life predicted from the decay equation described under "Experimental Procedures." The theoretical EhPgp5
mRNA half-life was 1.2 h in the trophozoites of clone C2,
2.7 h in C2(90), and increased to 5.6 h in C2(225) (Table
I).
EhPgp5 mRNA Presents 3'-UTR Heterogeneity--
To determine
whether the distinct EhPgp5 mRNA half-lives observed in
the trophozoites of clones C2, C2(90), and C2(225) could be influenced
by changes in the 3'-UTR length, we performed RNA protection assays
with S1 nuclease (Fig. 3A).
Three RNA-DNA-protected fragments were found at nt 118, 156, and 189 downstream of the UAA stop codon of the EhPgp5 mRNA in
all C2 clones (Fig. 3A, lanes 2-4). These
fragments, corresponding to different EhPgp5 3'-UTR variants, were denoted as PSII118, PSIII156,
and PSIV189, respectively. Interestingly, clone C2(225)
showed an extra protected fragment at the nt 19 (PSI19)
(Fig. 3A, arrow). The protected fragments
detected were analyzed by densitometry (Fig. 3B). The total
pixels obtained from the transcript species in clone C2(225) were taken
as 100%, and those from C2(90) and C2 clones were 53 and 47%,
respectively. In clone C2, the PSIV189,
PSIII156, and PSII118 variants showed levels of
26, 23, and 31 pixels, respectively (Fig. 3C). In clone
C2(90), PSIV189 and PSIII156 appeared in
similar amounts (36 and 34 pixels), whereas PSII118 gave
only 21 pixels (Fig. 3D). In clone C2(225), the transcript PSI19 was 40% of total variants (68 pixels), whereas
PSIV189, PSIII156, and PSII118gave
43, 39, and 23 pixels, respectively (Fig. 3E). In clone
C2(225), the PSIII156 and PSIV189 species
increased almost 2-fold with respect to PSII118, whereas
PSI19 was 3.3-fold, suggesting that they could be more
stable (Fig. 3E).
The presence of various polyadenylation sites suggested that mRNA
variants could influence the EhPgp5 mRNA steady-state
levels, as has been reported for other cells (38-40). Differences in
sequence at the 3'-UTR could be involved in the EhPgp5
mRNA stability and polyadenylation site selection. The comparative
analysis of the first 189 bases in the 3'-UTR EhPgp5 DNA
showed that 3'-UTR sequences were identical in clones A, C2 (Fig.
4), and C2(225) clones (data not shown),
indicating that differences in sequence do not account for the
EhPgp5 mRNA half-life or for the polyadenylation site choice. The EhPgp5 3'-UTR is 85% AU-rich, and it presents
several putative consensus binding sequences for regulatory proteins. There are five putative consensus polyadenylation signals (PS) with the
UA(A/U)UU sequence described for E. histolytica (41) at nt
1, 17, 98, 127, and 174 (Fig. 4, open boxes). We also found several eukaryotic 3'-UTR elements including the canonical
polyadenylation signal (AAUAAA) at nt 90 (Fig. 4, shadowbox)
and three putative AU-rich elements (AUREs) conformed by the conserved
AUUUA sequence (42) at nt 19, 76, and 99 downstream from the UAA codon
(Fig. 4, ellipses). In addition, two polypyrimidine tracts
(Py) were detected at nt 31-44 and 142-159 (Fig. 4,
discontinuous underlined) and a consensus cytoplasmic
polyadenylation element (CPE), (UUUUUAU) at nt 146-154 (Fig. 4,
continuous underlined). The CPE seems to promote cytoplasmic
polyadenylation in eukaryotic cells (43). Alignment of the
EhPgp5 3'-UTR full-length with other mdr 3'-UTR sequences revealed a high divergence between 3'-UTRs mdr
gene family members (data not shown).
A Specific RNA-Protein Complex Was Detected at the EhPg5 3'-UTR
mRNA--
Stability of mRNA is also regulated through
site-specific binding of cytoplasmic proteins to consensus sequences at
the 3'-UTR mRNA. The presence of putative EhPgp5
mRNA binding regulatory proteins was investigated by RNA
electrophoretic mobility shift assays using the PSI19,
PSII118 PSIII156 and PSIV189
fragments as RNA probes (Fig.
5A). The results showed that
PSIII156 and PSIV189 transcripts were able to
form a RNA-protein complex with CE from C2, C2(90), and C2(225) clones
(Fig. 5, B-D, lanes 2 and 3).
RNA-protein complex was specifically competed by a 350-fold molar
excess of the same unlabeled transcript, but they were maintained in
the presence of tRNA, used as nonspecific competitor (Fig. 5,
E and F, lanes 3 and 4).
The intensity and migration of the RNA-protein complex was similar in
all clones (Fig. 5, B-F). In contrast, we did not find any
RNA-protein complex when we used PSI19 and
PSII118 fragments (Fig. 5, B-D, lanes
4 and 5).
To delimitate the region in which the RNA-protein complex was formed,
we carried out cross-competition experiments using the PSIV189 transcript as labeled probe and the
PSI19, PSII118, and PSIII156 RNAs
as competitors. In the three clones the complex formed in the
PSIV189 region was specifically competed by the same probe
and by PSIII156 (Fig. 6,
A-C, lanes 5 and 6) but not by
PSI19 and PSII118 RNA fragments, as expected
(Fig. 6, A-C, lanes 3 and 4). These results suggest that RNA-protein interaction takes place in a region of
38 nt (nt 119-156), which is shared by PSIII156 and
PSIV189 fragments. This region contains a PS (nt 127-131)
and a 15-nt Py (nt 142-156) sequences, including a CPE (nt 146-154)
motif, which could be targets for regulatory RNA-binding proteins
(43-46).
The EhPgp5 mRNA Poly(A) Tail Is Longer in C2(225)
Trophozoites--
Poly(A) tail is an important modulator of mRNA
turnover, and its length is subjected to cellular control throughout
the life span of the mRNA (25). We investigated the poly(A) tail
length of EhPgp5 mRNA from trophozoites of clones C2,
C2(90), and C2(225) by LM-PAT (35), as described under "Experimental
Procedures." We observed three well defined bands corresponding to
the PSII118, PSIII156, and PSIV189
predicted transcripts plus 100 bp of the EhPgp5 open reading frame, respectively (Fig. 7A).
In these assays, we could not amplify the PSI19 variant,
probably because EhPgp5 PSI19 transcript has a
very short poly(A) tail. The identity of the amplified products was
confirmed by Southern blot hybridization with a DNA probe containing
the last 100 bp of the open reading frame and the first 19 nt of the
EhPgp5 mRNA 3'-UTR (Fig. 7B). The signal
appeared as a smear ranging from 218 to 300 nt in mRNA from
trophozoites of clones C2 and C2(90) (Fig. 7B).
Interestingly, in clone C2(225) the hybridization signal showed a
longer smear spanning from 218 to 500 nt. The same membrane hybridized
with the actin 3'-UTR probe gave no signal (Fig. 7C). In
contrast, in the actin control assays, we detected a defined 130-bp
band corresponding to actin 3'-UTR (~30 nt) plus 99 bases of the
3'-actin open reading frame and a short smear spanning 130-150 nt in
all clones (Fig. 7, D and E). The actin control
membrane gave no signal with the EhPgp5 3'-UTR probe (Fig.
7F).
These LM-PAT patterns represent an enlargement of the poly(A) tail
length of the EhPgp5 mRNA in the trophozoites of clone C2(225) or, alternatively, a shortening in C2 and C2(90) cells, suggesting that changes in poly(A) tail length are involved in EhPgp5 mRNA half-life.
Previously, we demonstrated that the EhPgp5 gene is
overexpressed in E. histolytica trophozoites grown in the
presence of high drug concentration (30). Transcriptional factors
participate in the EhPgp5 gene promoter activation (11).
Results presented here show novel evidence that post-transcriptional
EhPgp5 gene regulation occurs in the drug-resistant
trophozoites. EhPgp5 mRNA is more stable in trophozoites
grown in 225 µM emetine than in those grown in 90 µM or without drug (Fig. 2). Additionally, the EhPgp5 mRNA 3'-UTR length is heterogeneous (Fig.
3A), which may influence the mRNA half-life. The
PSIII156 and PSIV189 mRNA variants
augmented when the emetine dose was increased (Fig. 3,
A-E). Their predicted secondary structure suggests that
they have exposed a Py tract and a CPE motif (data not shown).
Furthermore, a RNA-binding protein complex was detected in their 39-nt
shared region (Figs. 5 and 6). In other organisms, polypyrimidine
tract-binding proteins have been involved in splicing and stability
control of the mRNA, whereas CPE-motif interacting proteins target
specific mRNAs to cytoplasmic polyadenylation, producing the
translational activation of the transcripts (43-46). Interestingly,
the EhPgp5 mRNA presents a longer poly(A) tail in clone
C2(225) (Fig. 7), and it is well known that large poly(A) tails give
higher stability to mRNA and promote a more efficient translation
(39). Emetine stress could affect the expression of many factors,
including the polyadenylation/deadenylation proteins involved in the
poly(A) tail length control.
mRNA half-life and translation are linked in ways that are not
completely understood. In cells exposed to translation inhibitors some
mRNAs are stabilized in several ways, including alterations in
polyadenylation rates (22). For example, in mammalian cells, cycloheximide prolongs c-myc mRNA half-life by slowing
the deadenylation process but does not promote degradation of the
mRNA body once deadenylation is being completed (47). The
heterogeneity of the EhPgp5 transcripts is explained by the
alternative usage of several polyadenylation signals detected in the
3'-UTR (Fig. 4), as has been well documented for other systems,
including other mdr genes (40, 48). Mouse mdr1a
mRNA shows length variations at both 5' and 3' ends, and mRNA
variants have very large 3'-UTRs, which are differentially
overexpressed in multidrug-resistant cell lines (48). Interestingly,
the EhPgp5 mRNA also presents heterogeneity in the 5'
end of trophozoites of clones C2 and C2(225) (11). All these data
indicate that EhPgp5 gene is a complex transcriptional unit
whose regulation produces multiple transcript sizes at the 3' and 5' ends.
Emetine partially inhibits protein synthesis in trophozoites of clone
C2(225) (data not shown). This could induce a stabilizing mRNA
effect (22, 49) affecting certain EhPgp5 transcript
variants. The PSIV189, PSIII156, and
PSII118 mRNA variants were detected in all clones,
whereas PSI19 was observed only in clone C2(225).
PSI19, PSIII156, and PSIV189
transcripts were more abundant in the trophozoites of clone C2(225). PSI19 transcript, which has a very short poly(A) tail
length or is not polyadenylated, was almost 2-fold the amount of
PSIII156 and PSIV189 in clone C2(225) (Fig. 3).
It is possible that the expression of undetermined factors linked to
the emetine effect and whose function could be independent of the
poly(A) tail contribute to the PSI19 transcript stability.
However, additional experiments are required to confirm this hypothesis.
AURE motifs have been involved in destabilization of mRNAs with a
short half-life (42). However, Prokipcak et al. (16) find
that AUREs at the 3'-UTR of human MDR1 mRNA is an
inefficient promoter of mRNA decay, which suggests that
AURE-dependent mRNA stability regulation may not
operate in certain cases, such as the MDR1 and
EhPgp5 mRNAs. This assumption is supported because under
the experimental conditions reported here, we did not detect any
RNA-protein complex in the AURE motifs present in the EhPgp5 3'-UTR mRNA, suggesting that they do not act as
cis-regulatory elements in the EhPgp5 mRNA
half-life control.
We found a RNA-protein complex in the proximity of the Py tract in the
PSIII156 and PSIV189 transcripts (Figs. 5 and
6) that may contribute to their stability in C2(225) cells. However,
the same complex was also detected in clones C2 and C2(90), suggesting
that other factors present only in clone C2(225) are required to
stabilize certain EhPgp5 mRNA variants. The identity of
the 3'-UTR EhPgp5 mRNA-interacting protein(s) detected
here remains to be elucidated. Interestingly, the
EhPgp5 mRNAs from trophozoites of clone C2(225) present
longer poly(A) tails than those from C2 and C2(90) cells, suggesting that polyadenylation and deadenylation events, occurring at different rates, could be affecting the EhPgp5 mRNA half-life.
Our working hypothesis assumes that trophozoites of clone C2 grown
without emetine have some factors that maintain short poly(A) tails,
which may contribute to a shorter EhPgp5 mRNA half-life (Fig. 8). Some of these factors could be
affected by emetine in C2(225) cells, and emetine-responsive factors
could both induce an enhanced polyadenylation of EhPgp5
mRNAs. The expression and activity of some proteins involved in 3'
to 5' exonucleolytic mRNA degradation and polyadenylation may also
be participating in the longer EhPgp5 mRNA half-life in
C2(225) cells.2 Hence, the
putative role of other 3' end processing and
polyadenylation/deadenylation factors cannot be discarded.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
reverse transcriptase (Invitrogen), and 40 units of SUPERase-in ribonuclease inhibitor (Ambion). This mixture was
incubated at 42 °C for 1 h. To remove the excess RNA
template, 2 units of RNase H (Amersham Biosciences) were added,
and the mixture was incubated at 37 °C for 15 min. Quantitative
multiplex PCR for EhPgp5 and actin cDNAs were performed
with 1/5 volume of the reverse transcription mixture, 10 mM each dNTPs, 5 mM MgCl2, 2.5 units of Taq DNA polymerase (Invitrogen), and the
EhPgp5 (5'-GTAGGAGGTGCAGTATTTCC-3') sense and
(5'-CCATCCTATTTCTTGTTTGAC-3') antisense internal primers (30). The
actin (5'-AGCTGTTCTTTCATTATATGC-3') sense and
(5'-TTCTCTTTCAGCAGTAGTGGT-3') antisense internal primers (31) were used
in the same sample as an internal control. PCR was done in 22 cycles at
95 °C for 30 s, 52 °C for 35 s, 72 °C for 90 s,
and a final extension step at 72 °C for 7 min. Amplified products
were separated by 6% PAGE.
-32P]dATP (PerkinElmer Life
Sciences)-labeled probe corresponding to the last 100 bp of the
EhPgp5 coding region and 597 bp of the EhPgp5
3'-UTR genomic sequence. The probe was PCR-amplified from the P4
plasmid, which contains the last 1466 bp of the
EhPgp5-coding region and ~1500 bp of its 3'-UTR genomic
sequence (7), using the EhPgp5-3'-UTR-S
(5'-AAAATAGTAGAACAAGGA-3') sense and EhPgp5-3'-UTR-AS (5'-CGAACAAAGGCTTAAA-3') antisense primers. Amplified
EhPgp5-3'-UTR fragment was sequenced in a ABI PRISM
automatic sequencer. Purified DNA probe was heat-denatured at 95 °C
for 15 min and cooled at 4 °C for 3 min. Then, an RNA aliquot was
added, and immediately, the DNA/RNA hybrid mixture was co-precipitated
with ethanol at
20 °C overnight. A DNA/RNA pellet was collected by
centrifugation, air-dried, and resuspended in 20 µl of S1 nuclease
hybridization buffer (80% deionized formamide, 40 mM MOPS,
pH 7, 400 mM NaCl, and 1 mM EDTA). The DNA/RNA
hybrids were fully denatured at 85 °C for 10 min and subsequently
incubated at 42 °C overnight. Then, samples were 10-fold diluted and
treated with 1250 units of S1 nuclease (Invitrogen) at 37 °C for
1 h in the reaction buffer (300 mM sodium acetate, pH
4.6, 1 mM NaCl, and 10 mM zinc acetate). At the
same time, we performed a sequencing reaction of the EhPgp5 3'-UTR using the EhPgp5-3'-UTR-S primer. The sequencing
products and RNA fragments protected of the S1 nuclease digestion were resolved through denaturing 8% PAGE at room temperature, vacuum-dried, and visualized in a PhosphorImager apparatus.
) phagemid (pBS)
(Stratagene), which contains the T7
phage promoter. The
PSI19, PSII118, PSIII156, and
PSIV189 DNA fragments that contain the last 100 bp of
EhPgp5 open reading frame and 19, 118, 156, and 189 bp of
the 3'-UTR, respectively, were PCR-amplified using 1 unit of Deep Vent
DNA polymerase (New England Biolabs) and the P4 plasmid (7) as
template. The primers used were EhPgp5-3'-UTR-S sense and
the PSI-AS (5'-TTAATATTTATATGAATTA-3'), PSII-AS
(5'-TTATTATGAATGATAAATA-3'), PSIII-AS (5'-AAAGAATAAAAACAAACT-3'), and
PSIV-AS (5'-TATCATATAACAATTAAA-3') antisense primers. Amplified products were directionally cloned into the BamHI and
XhoI sites of pBS plasmid and sequenced in a ABI PRISM
automatic sequencer. Recombinant plasmids (1 µg each) were linearized
with XhoI restriction enzyme and in vitro
transcribed with T7 RNA polymerase using 1 mM each NTP, 100 mM dithiothreitol, and 40 units of recombinant RNasin
ribonuclease inhibitor (Invitrogen) at 37 °C for 1 h according to the manufacturer protocol (Promega). Labeled EhPgp5
3'-UTR RNA fragments were produced by the addition of
[
-32P]UTP (3000 Ci/mmol) (PerkinElmer Life Sciences)
in the synthesis reaction. Finally, RNase-free DNase (10 units) was
added, and the mixture was incubated at 37 °C for 15 min to remove
DNA templates. Labeled RNA probes were purified by Sephadex G50 column
filtration. EhPgp5 3'-UTR transcripts length was determined
by 12% denaturing PAGE.
-32P]UTP-labeled probes and 60 µg of CE were
separately incubated in the binding buffer (10 mM HEPES, pH
7.9, 40 mM KCl, 1 mM dithiothreitol, 4 mM MgCl2, 4 mM spermidine, 5%
glycerol) at 4 °C for 15 min in 20 µl of final volume.
Subsequently, RNase A + T1 (10 µg + 20 units) (Sigma)
were added to the mixture and incubated at room temperature for 15 min.
Then, heparin (5 mg/ml) was added, and the mixture was incubated for an
additional 10 min. For competition assays, we used a 350-fold molar
excess of the PSI19-, PSII118-,
PSIII156-, and PSIV189-unlabeled probes.
RNA-protein complexes were resolved at 130 V for 4 h on
pre-electrophoresed 6% nondenaturing PAGE. Gels were vacuum-dried, and
RNA-protein interactions were detected by scanning in a PhosphorImager apparatus.
-32P]dATP-labeled probes.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of actinomycin D on RNA synthesis in
the E. histolytica trophozoites of clones A and
C2. A, growth curve of the trophozoites of clone C2
treated with actinomycin D. B, effect of actinomycin D on
RNA synthesis in the trophozoites of clones A and C2. Actinomycin
D-treated trophozoites were incubated with
[3H]UTP (10 µCi/mmol) for 2 h, and incorporation
was measured in 20 µg of total RNA. Incorporation values at
t0 were taken as 100% for each clone.
Bars represent the average of three duplicate experiments.
C, formaldehyde-agarose gel (1.2%) showing the recombinant
RNA obtained from actinomycin D-treated trophozoites of
clones A (upper panel) and C2 (lower panel) at
the indicated times. DMSO, dimethyl sulfoxide.
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Fig. 2.
Determination of the EhPgp5
mRNA half-life. A-C and G,
ethidium bromide-stained gels showing the RT-PCR products obtained at
different times after actinomycin D transcriptional blockage.
MW, molecular weight. D-F, densitometric
analysis of RT-PCR products in A-C. H,
semilogarithmic plot of the EhPgp5 and actin mRNA levels
quantified by densitometry in A-C. The graphics showed the
results of a representative assay of three independent experiments.
Actin: , C2;
, C2(90);
, C2(225). EhPgp5:
, C2;
, C2(90);
, C2(225).
Theoretical and experimental EhPgp5 mRNA half-life in E. histolytica trophozoites of clone C2 grown at different emetine
concentrations
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Fig. 3.
Determination of the EhPgp5
mRNA polyadenylation sites. 20 µg of total RNA from
trophozoites of clones C2, C2(90), and C2(225) were hybridized with a
genomic EhPgp5 3'-UTR probe and then digested with S1
nuclease. A, 8% PAGE analyzed products alongside
EhPgp5 3'-UTR sequence ladder. Numbers at the
right indicate the size of protected fragments. Lane
1, tRNA negative control hybridization; lane 2, clone
C2; lane 3, clone C2(90); lane 4, clone C2(225).
The arrow shows the extra polyadenylation site detected in
trophozoites of clone C2(225). TAA indicates the translation stop codon
position. B, representative data of the analysis by
densitometry of bands in A. Total pixels obtained from the
transcript species in clone C2(225) were taken as 100%, and those from
C2(90) and C2 clones were expressed in relation to it. C-E,
graphic representations of the relative amounts of each transcript
variant in all clones. These assays were performed three times, and
relative transcript values were reproducible.
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Fig. 4.
Sequence of the 189 bp EhPgp5
3'-UTR of clones A and C2. Arrows show the
position of the four polyadenylation sites mapped (PSI19,
PSII118, PSIII156, and
PSIV189). E. histolytica consensus
polyadenylation signals (UA(A/U)UU) are in open boxes.
Eukaryotic poly(A) signal (AAUAAA) is indicated by a
shadowbox. Continuous and discontinuous
underlines denote the CPE motif and the polypyrimidines tract,
respectively. Ellipses denote the AU-rich elements (AUUUA).
Numbers at the right are relative to the
translation stop codon (position 1).
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Fig. 5.
RNA-protein complex formation in the
EhPgp5 3'-UTR mRNA. A, schematic
representation of the putative cis-acting elements present
in the 3'-UTR of the EhPgp5 fragments used for RNA
electrophoretic mobility shift assays. EPS, eukaryotic
polyadenylation signal; UAA, stop codon. ORF, open reading frame.
B-D, [ -32P]UTP-labeled PSI19,
PSII118, PSIII156, and PSIV189
probes were incubated with CE from trophozoites of the different
clones, as described under "Experimental Procedures." Lane
1, free PSI19; lane 2,
PSIII156; lane 3, PSIV189;
lane 4, PSI19; and lane 5,
PSII118 probes. E and F, competition
experiments. The PSIII156 (E) and
PSIV189 (F) riboprobes were incubated with CE
from the trophozoites in the presence of free probe (lane
1), no competitor (lane 2), specific competitor
(SC; lane 3) (350-fold molar excess of homologous
unlabeled fragments), and unspecific competitor (UC;
lane 4) (350-fold molar excess of tRNA). Arrows
denote RNA-protein complex.
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Fig. 6.
Competition of the RNA-protein complex formed
in the PSIV189 EhPgp5
transcript. [ -32P]UTP-labeled full-length
PSIV189 probe was incubated with CE (lanes 2-6)
from trophozoites of the different clones in the presence of different
unlabeled probe competitors. Lane 1, free probe; lane
2, no competitor; lane 3, PSI19; lane
4, PSII118; lane 5, PSIII156;
lane 6, PSIV189 probes. Arrows show
the RNA-protein complex.
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Fig. 7.
LM-PAT of EhPgp5 and actin
mRNAs. Ethidium bromide-stained agarose gels (1.5%) of the
EhPgp5 (A) and actin (D)
LM-PAT-amplified products using RNA from trophozoites of the three
clones. (B, C, E, and F,
PhosphorImager scanning of A and D membranes
hybridized with (B and F) EhPgp5- and
(C and E) actin-specific 3'-UTR probes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Working model for EhPgp5
mRNA expression in C2 cells. A, an enhanced
transcription of EhPgp5 gene occurs in trophozoites of clone
C2(225) due to the effect of emetine on the transcriptional factors.
ORF, open reading frame. B, in nuclei and
cytoplasm, mRNA processing and decay mechanisms influence the
EhPgp5 mRNA half-life. C, trophozoites of
clone C2(225) have putative emetine-responsive factors and
polyadenylation proteins, which could promote an efficient
polymerization of poly(A) tails of certain EhPgp5 mRNA
variants, resulting in a longer poly(A) tail and increased mRNA
stability. CPE- and Py-interacting proteins, present in both clones,
could promote in clone C2(225)-specific readenylation of
EhPgp5 mRNA due to the expression of emetine-responsive
factors, which may also participate in the blockage of the progress of
the putative 3'-5' mRNA degradation machinery. Deadenylase activity
in trophozoites of clone C2 could be enhanced, resulting in shorter
poly(A) tails and diminished EhPgp5 mRNA half-life.
Wave lines denote the EhPgp5 mRNA, and the
filled circle represents the UAA stop codon. Potential Py-
and CPE-interacting proteins are indicated as a RNA-protein
complex.
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ACKNOWLEDGEMENTS |
---|
We express gratitude to Drs. Lorena Gutiérrez and Rosa Maria Del Angel for critical reading of this manuscript. Our thanks go also to Alfredo Padilla for help with the artwork.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Howard Hughes Medical Institute (to E. O.) and by Consejo Nacional de Ciencia y Tecnologia (México) and European Community grants.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. Tel.:
52-55-5747-3800 (ext. 5650); Fax: 52-55-5747-7108; E-mail:
esther@mail.cinvestav.mx.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M211757200
2 C. López-Camarillo, J. P. Luna-Arias, L. A. Marchat, and E. Orozco, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: MDR, multidrug resistance; PGP, P-glycoprotein; Py, polypyrimidine tracts; CPE, cytoplasmic polyadenylation element; UTR, untranslated region; RT, reverse transcriptase; MOPS, 4-morpholinepropanesulfonic acid; CE, cytoplasmic extracts; LM-PAT, ligase-mediated poly(A) test; nt, nucleotide(s); PS, polyadenylation signals; AURE, AU-rich element.
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