From the Instituto de Investigaciones Biotecnológicas, Instituto Tecnológico de Chascomús, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de General San Martín, 1650 San Martin, Provincia de Buenos Aires, Argentina
Received for publication, December 5, 2000, and in revised form, February 5, 2001
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ABSTRACT |
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Post-transcriptional regulatory mechanisms have
been suggested to be the main point of control of gene expression in
kinetoplastid parasites. We have previously shown that
Trypanosoma cruzi SMUG mucin mRNA steady-state level is
developmentally regulated by post-transcriptional mechanisms, being
stable in the epimastigote insect vector stage, but unstable in the
trypomastigote infective stage of the parasite. Its turnover is
controlled by an AU-rich element (ARE) localized in the 3'-untranslated
region, since a reporter gene lacking this sequence was stable in the
trypomastigote stage (Di Noia, J. M., D'Orso, I., Sanchez,
D. O., and Frasch, A. C. (2000) J. Biol.
Chem. 275, 10218-10227). Here, we show by gel mobility shift
assay that the 44-nt ARE sequence interacts with a set of
stage-specific AU-rich element RNA-binding proteins (ARE-BPs). The
epimastigote stage AU-rich element RNA-binding protein, named E-ARE-BP,
and the trypomastigote stage ARE-BPs, named T-ARE-BPs, are efficiently
competed by poly(U). UV cross-linking analysis showed that E-ARE-BP has
an apparent molecular mass of 100 kDa and is different from the
45-50-kDa ARE-BPs present in other stages of the parasite.
Transfection experiments allowed the identification of a novel
cis-element that might be responsible for a positive effect
on mRNA stability. It is a G-rich element, named GRE, composed by
two contiguous CGGGG pentamers. The factors that recognize GRE were
different from the ones that bind to ARE, in both molecular masses and
subcellular localization. Thus, ARE and GRE are functionally different
cis-elements, which might regulate mucin expression
throughout the parasite life cycle.
Kinetoplastid parasites control gene expression at the level of
mRNA maturation by post-transcriptional mechanisms (reviewed in
Refs. 2 and 3). In vivo treatment of parasites with protein synthesis
inhibitors induces an accumulation (9) or a decrease in the mRNA levels of some transcripts (1), and this effect is not due to an
increase or a reduction in transcriptional levels, respectively. Therefore, these results point to the presence of labile factors, affected by protein synthesis inhibitors, that might be negative or
positive regulators of mRNA maturation. However, the mechanisms of
interference in pre-mRNA processing, unbalanced nucleo-cytoplasmic transport, or unusual mRNA stability control processes remain to be
identified. It is known that both 5'- and 3'-untranslated regions
(UTRs)1 are responsible for
stabilization/destabilization mechanisms, up- or down-regulating
mRNA levels in a developmentally regulated manner (10, 11). In
transient and stable parasite transfection experiments, the 3'-UTRs of
some mRNAs were found to influence the expression of a reporter
gene in a stage-specific manner (1, 10, 11). The way in which the
3'-UTR differentially influence the mRNA steady-state levels is
still unknown. Furthermore, few cis-elements responsible for
these post-transcriptional regulatory mechanisms have been defined
(12-14).
Several cis-elements and trans-acting factors
controlling mRNA stability have been characterized in higher
eukaryotes (15, 16). A well known example is the case of AU-rich
elements or AREs, cis-sequences localized in the 3'-UTR of
short-lived mRNAs, such as proto-oncogenes and cytokines (17).
These elements are recognized by different positive or negative
RNA-binding proteins, like HuR and AUF-1/heterogeneous nuclear
ribonucleoprotein D, respectively (18-20), causing rapid changes in
mRNA stability. Another example is the ribonucleoprotein complex
associated with human Trypanosoma cruzi, the protozoan parasite agent of Chagas
disease, is covered by a dense mucin coat (24), at least in two of its
developmental stages. Mucins are highly O-glycosylated proteins having relevant roles in cell protection and in cell-cell interactions, especially in immune cell migration in vertebrate cells
(25). Mucins from T. cruzi were classified into two
different protein families that differ between parasite stages. The
form of the parasite present in the insect vector, epimastigote,
expresses a small mucin family named TcSMUG (35-50 kDa) whose core
proteins are encoded in about 70 different genes (1), while the forms of the parasite present in the mammalian host, bloodstream
trypomastigotes, have larger mucins encoded by 500 different genes
(26). Developmentally regulated expression of these mucins in the
different parasite stages is relevant because they might accomplish
different functions related with parasite survival (27).
We have previously demonstrated that a 44-nt ARE sequence within the
3'-UTR of SMUG mucin family was a destabilizing cis-element acting in a stage specific manner (1). These results suggest that
different trans-acting factors might bind mucin transcripts in vivo, and selectively regulate its mRNA stability
throughout parasite development. We have now identified a novel G-rich
element, named GRE, which might be responsible for a stage-specific
stabilization of SMUG mRNA family in the epimastigote form of the
parasite. Transfection experiments show that GRE and ARE sequences have opposite functions in terms of mRNA stabilization in the different stages of the parasite and are specifically recognized by
trans-acting factors, some of them being developmentally
regulated during the trypanosome life cycle.
Parasite Cultures and Drug Treatments--
Trypanosoma
cruzi CL-Brener cloned stock (28) was used. Different forms of the
parasites were obtained as described previously (29). Purity of the
different parasite forms was determined by conventional microscopy and
was at least 95%. Epimastigote cultures were taken in logarithmic
growth phase at a cell density of 3 × 107/ml and
treated with actinomycin D (ActD) (Sigma), at a final concentration of 10 µg/ml, which is known to inhibit transcription in
trypanosomatids (12, 30). Aliquots were taken at different times after
addition of the inhibitor. Cycloheximide (Sigma) was used at a final
concentration of 50 µg/ml (31). Parasite viability was confirmed by
microscopy at every time point of the experiments. Culture aliquots
were harvested by centrifugation, washed with phosphate-buffered
saline, and frozen at Chloramphenicol Acetyltransferase (CAT) Assay--
An equal
number of parasites from each transfected population was harvested,
washed once with 0.25 M Tris-HCl (pH 8), and cellular
extracts were prepared by four freeze-thaw cycles and heat
inactivation. Cell lysates were assayed for CAT activity as described
previously (32). Reactions were conducted for 1 h at 37 °C with
cellular extracts prepared from 107 parasites. This time
was previously adjusted to fit within the linear range of the assay.
Conversion of [14C]chloramphenicol to acetylated forms
was analyzed by thin layer chromatography and quantified by densitometry.
DNA Constructions and Parasite Transfections--
The
chloramphenicol acetyltransferase (cat) gene, the complete
TcSMUG intergenic region, and the SMUG-L and SMUG-L
Each DNA fragment was cloned in the pTEX vector (33), kindly provided
by Dr. J. M. Kelly (London School of Hygiene and Tropical Medicine, London, United Kingdom). Transfections were carried out as
described previously (1). The neo resistance gene was used
for selection and as an internal control of transfection levels since
it is transcribed polycistronically from the same promoter (33). The
polyadenylation site of the cat mRNA was determined by reverse
transcription-PCR using the oligonucleotide anchor d(T) (5'-
GCGAGCTCCGCGGCCGCG(T)18-3') using the Superscript II enzyme
(Life Technologies, Inc.). PCR was performed on first strand product
using CAT/se (5'-gggATGGAGAAAAAAATCACTGGATATA-3') and an
oligonucleotide with the anchor sequence of anchor d(T). The products
were cloned in pGEMT-Easy (Promega, Madison, WI) and sequenced.
In Vitro Transcription--
All plasmids for in vitro
transcription were constructed as follows. Complementary
oligonucleotides, corresponding to the sense and antisense strands of
the RNAs transcribed, were annealed and cloned into the
EcoRI and HindIII sites of the vector pBS Protein Extract Preparation and Subcellular
Fractionation--
For total protein extract preparation, parasites
were resuspended in lysis buffer (0.75% CHAPS detergent, 1 mM MgCl2, 1 mM EGTA, 5 mM Analysis of RNA-Protein Interactions--
Binding reactions were
performed with 10 µl (3 µg/µl) of trypanosome total extract
(prepared as above), 10,000 cpm of RNA probe, 10 mM
Tris-HCl (pH 7.6), 5% glycerol, 100 mM KCl, 5 mM MgCl2, 1 µg/ml bovine serum albumin, 500 ng/µl tRNA (Sigma) in a 20 µl final volume. The incubation time was
10 min at 25 °C. Heparin was added at a concentration of 1 µg/ml.
Each reaction was loaded directly onto a 7% acrylamide-bisacrylamide
(38:2), 0.5× TBE nondenaturing gel to perform an electrophoresis
mobility shift assay (EMSA). The gels were dried and exposed to film at UV Cross-linking Analysis--
32P-Labeled RNA was
incubated with a trypanosome total extract as described above. The
in vitro binding reaction was run on a 7%
acrylamide-bisacrylamide (38:2), 0.5× TBE native gel. The RNA-protein
complexes detected by exposing to films at 4 °C, cross-linked by UV
light irradiation (254 nm, 500 mJ/cm2), treated with RNase
T1, were cut from the gel and eluted with 0.1% SDS at 37 °C with
vigorous shaking. The cross-linked products were resolved by
electrophoresis on 10% SDS-PAGE, and the apparent molecular masses of
the proteins were determined with molecular size protein standards.
Northern Blot--
RNA was purified using TRIzol reagent
following the manufacturer's instructions (Life Technologies, Inc.).
Northern blots were carried out as described previously (38).
Zeta-Probe nylon membranes (Bio-Rad) were used for all blottings.
Probes were radioactively labeled with [ Both Positive and Negative cis-Elements within the 3'-UTR of SMUG
Mucin Family Regulate mRNA Stability and Translation
Efficiency--
TcSMUG mucin family was previously shown to be
post-transcriptionally regulated and an ARE within its 3'-UTR was found
to be responsible for a destabilizing mechanism acting in a
stage-specific manner (1). However, this mucin family is very stable in
the epimastigote stage, and the ARE motif was not responsible for this
selective mRNA stabilization (see below). We now searched for other
cis-elements required for mRNA stability and/or
translational control in this parasite stage. Five 3'-UTR deletion
mutants of the SMUG-L clone were constructed (Fig.
1A). Each mutant is deleted in
one of the blocks in which the mucin 3'-UTR is organized (1). The
complete construction consists of the cat gene flanked by both 5' and 3' intergenic regions of SMUG-L group, which contains sequences that ensure correct trans-splicing and
polyadenylation, cloned in the pTEX vector (33).
Half-life determinations of transcripts from the complete construct
(SMUG-L) and deletion mutants were carried out in the epimastigote form
of the parasite (Fig. 1), taking advantage of the presence of an
ActD-sensitive promoter in the pTEX vector. The transcript from the
complete construct SMUG-L had a half-life of about 70 min. Conversely,
SMUG-L
In order to determine if the different domains of the 3'-UTR also
influence expression at the translational level, the CAT activity from
control and deletion mutants was measured and the values obtained were
normalized to cat mRNA steady-state levels from each
construct (Fig. 2). Enzymatic activity
was expressed as the percentage of that obtained with the complete
construct SMUG-L. The value obtained with the parasite population
transfected with SMUG-L A Novel GRE Confers mRNA Stability in a Stage-specific Manner
and Is Functionally Different from the AU-rich Instability
Element--
The effect of the GRE deletion (SMUG-L
Analysis of the infective metacyclic trypomastigotes stage, derived
from differentiation of epimastigotes, also revealed differences in the
mRNA steady-state levels. Both SMUG-L and SMUG-L The 27-nt GRE That Confers mRNA Stability Specifically
Interacts with Different Nuclear and Cytoplasmic Complex-forming
RNA-binding Proteins--
The identification of this novel
cis-element involved in a mRNA stabilization process
allowed the searching for trans-acting factors able to
recognize G-rich sequences. The 27-nt GRE sequence was transcribed
in vitro as described under "Experimental Procedures" and used to perform RNA-protein binding reactions and EMSA. The SMUG-L-GRE RNA oligonucleotide revealed the same three
ribonucleoprotein complexes in all four parasite forms tested (Fig.
4A). As controls, no bands
corresponding to the G-complexes 1, 2, and 3 were observed after
incubation of SMUG-L-GRE RNA with RNase A or the protein extract with proteinase K (data not shown). To determine the apparent molecular masses of the proteins that compose the GRE-ribonucleoprotein complexes, a total protein extract from the epimastigote form of the
parasite was incubated with an excess of 32P-labeled
SMUG-L-GRE RNA oligonucleotide. The in vitro
binding reactions were run in a native polyacrylamide gel and after UV cross-linking, the complexes were treated as under "Experimental Procedures" and further electrophoresed in a 10% SDS-PAGE (Fig. 4B). G-complex 1 gave rise to a single band having an
apparent molecular mass of 80 kDa, while G-complexes 2 and 3 are
composed by several proteins with apparent molecular masses of
35, 39, and 66 kDa. The RNA-binding proteins that compose G-complex-2 are present in different abundance in the epimastigote total lysate. One low abundant protein of about 66 kDa is detected together with two
highly abundant factors of 35 and 55 kDa (Fig. 4B).
Competition experiments were conducted to further characterize the
sequence specificity of all complexes formed. Each of the four
homoribopolymers was used to compete with the SMUG-L-GRE RNA oligonucleotide in an in vitro binding reaction. Poly(G)
selectively blocks the assembly of two ribonucleoprotein complexes,
G-complex 1 (smaller band) and G-complex 2 (Fig. 4C). This
result is in agreement with the G-rich nature of the
cis-element, used in the in vitro binding
reaction. G-complex 1 is effectively competed out with a molar excess
of 10-fold, whereas the formation of G-complex 2 partially disappeared
at a molar excess of 1000-fold. This result could be due to differences
in the concentration of protein-forming complexes in the epimastigote
lysate and is also suggested by the UV cross-linking analysis (Fig.
4B), where G-complex 1 is barely detectable comparing with
the amount of proteins forming G-complex 2. Conversely, complex 3 was
not efficiently competed by any homoribopolymer and, thus, might be unspecific.
The minimal size of the SMUG-L-GRE RNA element recognized
by the proteins forming G-complexes 1 and 2 was analyzed. The RNA sequence was divided into two separate sequences: (a)
SMUG-L-GRE-1, with the sequence GGACGGGGCGGGGC; and
(b) SMUG-L-GRE-2, which presents a CG-rich
content, GCGCGUGCGCCG (Fig.
5A). The
SMUG-L-GRE-1 RNA is sufficient to interact with both
trans-acting factors (Fig. 5B). This result
suggest that the minimal sequence for G-complex 1 and 2 formation is
the first half of the element, which is composed of two contiguous
CGGGG pentamers. G-complex 1 is localized in the cytoplasm, whereas
G-complex 2 is equally distributed in both compartments, nucleus and
cytoplasm (Fig. 5B).
The 44-nt AU-rich Instability Element Interacts with
Stage-specific, Developmentally Regulated, RNA-binding
Proteins--
The 44-nt AU-rich cis-element was important
in conferring mRNA instability in a stage-specific manner (1) (Fig.
3). Therefore, to know if the RNA-binding proteins that recognized
in vitro this element, named here SMUG-L-AU, are
developmentally regulated, protein extracts from the four different
parasite stages were incubated with the RNA template in an in
vitro binding reaction. The complexes formed were identified in a
native polyacrylamide gel (Fig.
6A). A stage-specific pattern
of RNA binding to this motif was observed. In the epimastigote stage,
an RNA-binding protein named E-ARE-BP, for epimastigote AU-rich element
binding protein, migrated much more slowly in the polyacrylamide native gel than the ribonucleoprotein complexes detected in the other three
parasite stages. To determine the apparent molecular masses of these
RNA-binding proteins, the total protein lysate of each parasite stage
was incubated with an excess of SMUG-L-AU RNA probe and the
ribonucleoprotein-complexes identified in the EMSA were UV-cross-linked
and further electrophoresed in SDS-PAGE. The E-ARE-BP had an apparent
molecular mass of ~100 kDa. In contrast, the ARE-BPs range between 45 and 50 kDa (Fig. 6B). Both results, 1) the ARE deletion
affecting SMUG mucin mRNA stability (Fig. 3), and 2) the
developmentally regulated expression pattern of the RNA-binding proteins that recognized the ARE motif (Fig. 6, A and
B), point to a coordinated and stage-specific process during
the life cycle parasite development.
Competition experiments were carried out to further confirm the
specificity of the RNA-binding protein of the epimastigote form of the
parasite that recognized the 44-nt SMUG-L-AU RNA template. Results with the four homoribopolymers showed that E-ARE-BP is selectively competed by poly(U) (Fig.
7A) but not by the other three
homoribopolymers, as expected due to the U-rich nature of this element.
Unlabeled sense and antisense RNAs were also tested in competition
experiments (Fig. 7B). The addition of increasing amounts of
unlabeled sense SMUG-L-AU RNA to the reaction mixture resulted in a concentration-dependent reduction in the
formation of the ribonucleoprotein complex containing E-ARE-BP, whereas the addition of unlabeled antisense SMUG-L-AU RNA had
little effect on the formation of this complex. Trypomastigote ARE-BPs
(T-ARE-BPs) are also efficiently competed by poly(U) RNA, and not by
any other homoribopolymer (Fig. 7C). Additionally, we tested
the competition with unlabeled in vitro transcribed
SMUG-L-AU sense and antisense RNAs. The
SMUG-L-AU sense RNA, as was shown for the E-ARE-BP, abolished the binding of the ARE-BPs in a
concentration-dependent manner. This result confirmed that
the T-ARE-BPs selectively and specifically recognized the AU-rich
sequence of SMUG mRNAs (Fig. 7D) and that the U-rich
nature of the oligoribonucleotide is important for the binding.
Different Subcellular Localization of ARE RNA-binding
Proteins--
The presence of both AU and G-rich binding activities
was analyzed in a nuclear and cytoplasmic preparation of T. cruzi epimastigotes and trypomastigotes. Subcellular fractionation
was done as described under "Experimental Procedures." These
experiments showed that the E-ARE-BP is mainly cytosolic or that the
E-ARE-BP might recognize the SMUG-L-AU RNA only in the
cytoplasm and not in the nucleus (see "Discussion") (Fig.
8A). In contrast, the
45-50-kDa T-ARE-BPs are localized in similar amounts in both
compartments, nucleus and cytoplasm (Fig. 8C).
ARE-binding proteins of higher eukaryotes were shown to be associated
with polysomes, and this particular localization was due to a
translational regulatory mechanism conferred by those trans-acting factors (41-43). In a previous work, we
reported that the ARE motif positively regulates translation efficiency
in the epimastigote stage of the parasite (1), as is the case with the
ARE sequences in TNF-
We conclude that E-ARE-BP is mainly cytoplasmic and may be partially
associated to polysomes, whereas T-ARE-BPs are localized in both
compartments and may be nuclear-cytoplasm shuttling RNA-binding proteins.
In this work we have obtained evidence for the existence of novel
cis-elements localized in the 3'-UTR of SMUG mucins from T. cruzi that control both mRNA stability and
translation efficiency. In addition to the AU-rich element involved in
the selective mRNA destabilization of mucin transcripts in the
metacyclic trypomastigote stage of the parasite (Ref. 1 and this work),
new negative and positive cis-elements have now been
identified. First, a small GRE, composed of the first 27-nt downstream
stop codon and containing two contiguous CGGGG pentamers, functions as
a positive element only in the epimastigote stage of the parasite.
Second, deletion of another element in the construction SMUG-L Two functionally different cis-elements, ARE and GRE, were
identified. The ARE was involved in mRNA destabilization in the infective stage of the parasite, but not in the replicative
epimastigote stage, because mRNAs from SMUG-L and SMUG-L Cellular factors interacting with RNA motifs that regulate mRNA
stability have not been identified yet in trypanosomes. Evidence showing that GRE and ARE RNA sequences interact with different cellular
trans-acting factors has now been obtained (Figs. 4 and 6,
and summarized in the model of Fig. 9).
Three GRE-forming ribonucleoprotein complexes were detected. Two of
them, named G-complex 1 and G-complex 2, were specifically and
efficiently competed by poly(G) homoribopolymer (Fig. 4C).
G-complex 1 is formed by a single protein band whose apparent molecular
mass is 80 kDa, and G-complex 2 is composed of several factors whose
molecular masses were about 35, 39, and 66 kDa. This suggests that the
80-kDa protein of G-complex 1 directly recognized GRE sequence. In the
case of G-complex 2, the three proteins might also be involved in
protein-protein interactions. The presence of large complexes might
regulate mRNA expression in a coordinated way, depending on the
proteins that compose it or the protein-protein interactions that are
produced during the different stages of the parasite. Since the
presence of the ARE within SMUG-L 3'-UTR led to a rapid mRNA decay,
it is possible that a coordinated interaction between GRE-binding
proteins with ARE-BPs and/or other protein factors not identified yet
might determine the final mucin SMUG mRNA stability (Fig. 9).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Amanitin-sensitive RNA polymerase II from
trypanosomes transcribes large polycistronic units containing a number
of coding sequences (4). Transcriptional start sites have been
extremely difficult to detect; only two putative promoter regions were
described as transcriptionally void regions upstream from the actin and
Hsp70 genes (5, 6). The maturation of polycistronic RNA precursors to
render individual mRNA molecules is achieved by cleavage in the
intergenic region by a coupled processing of 5' end
trans-splicing and 3' end polyadenylation (7). Both
processes seem to depend on the recognition of polypyrimidine tracts
present in the intergenic regions (8), which acts as a bifunctional
element affecting RNA processing both upstream and downstream from
itself (7).
-globin mRNA (21). A cytidine-rich
(C-rich) segment within the 3'-UTR of
-globin is critical for
mRNA stability through the interaction with different
trans-acting factors that mediate this effect (22). However,
it has been shown that neither
CP1 nor
CP2 complex-forming
proteins can bind the C-rich element unless they are complexed with the
remaining non-poly(C)-binding proteins, such us AUF1/heterogeneous
nuclear ribonucleoprotein D (23). Thus, a protein implicated in
ARE-mediated mRNA decay is also an integral component of the
mRNA stabilizing
-complex.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C until RNA extraction.
AU constructs were amplified by PCR as described previously (1). All 3'-UTR deletions
were created by PCR and fused downstream from cat into the
HindIII and XhoI sites. The PCR primers contained
restriction enzymes sites to facilitate the subsequent cloning steps as
indicated. All the primers used to generate internal deletion mutants
of the 3'-UTR are listed below. For clone SMUG-L
GRE: 1-SE,
5'-cccaagcttTCGGTCTGGAGTCCCGTTGTG-3'; and 3'-AS,
5'-cggaattcGCACGCACAACCAAAGACACC-3'. For clone SMUG-L
1: 1+-SE,
5'-cccaagcttTGCGCCGCGTATATTTGAGC-3'. Two oligonucleotides containing the first 27 base pairs with a HindIII
restriction site: sense, 5'-agcttTGAGGACGGGGCGGGGCGCGTGCGCCGA-3'; and
antisense, 5'-agcttCGGCGCACGCGCCCCGCCCCGTCCTCAA-3', were annealed and
cloned rendering the complete clone pTEX-SMUG-L
1. For clone
SMUG-L
2: 2-SE, 5'-cggaattcTGCCCGTGCATTCTCTGCGAC-3'; and 2-AS,
5'-cggaattcAAATATACGCGGCGCAGGCGCC-3'. For clone SMUG-L
3: 3-SE,
5'-cggaattcGTGGGGGGAAGCGTCGGTGCG-3'; and 3-AS. For clone
SMUG-L
Sire: Sire-SE, 5'-cggaattcAGGCGAATATTTTTTTTCATCGG-3'; and
Sire-AS, 5'-cggaattcCCCCCGCGAACATCCCTTTCAC-3'.
(Stratagene, La Jolla, CA). Transcription of sense sequences was performed with 1 µg of HindIII-digested plasmids using T7
RNA-polymerase (Promega) in the presence of [
-32P]UTP
(800 Ci/mmol, PerkinElmer Life Sciences), 500 µM ATP,
CTP, and GTP. Antisense transcripts were synthesized with T3 RNA
polymerase. All transcripts were purified on a 8 M urea,
12% polyacrylamide gel and eluted overnight in RNA elution buffer (0.3 M NaOAc, 10 mM MgCl2, and 1 mM EDTA). After elution, RNAs were ethanol-precipitated and
resuspended in 50 µl of water. Preparative in vitro
transcription was done as described previously (34) and detected by UV shadowing.
-mercapthoethanol, 10 mM Tris-HCl (pH
7.6), and 10% glycerol) supplemented with protease inhibitors: 1 mM phenylmethylsulfonyl fluoride and 50 µM
E-64 (Sigma). After 30 min on ice, the extract was centrifuged at
19,000 rpm (SS-34 rotor) and the supernatant stored at
70 °C. For
subcellular fractionation, nuclear and cytoplasmic fractions were
prepared as described previously for another kinetoplastid parasite,
Crithidia fasciculata (35). Briefly, parasites were washed
twice in Buffer A (10 mM Tris-HCl (pH 7.6), 1.5 mM MgCl2, 10 mM KCl) and
resuspended in Buffer B (Buffer A plus 1 mM dithiothreitol, 1 mM EDTA, and 0.5% Nonidet P-40) in the presence of
protease inhibitors. After 20 min on ice and vortexing each 3 min, the preparation was centrifuged for 15 min at 5000 rpm. The supernatant containing the cytosolic fraction was mixed with an equal volume of
Buffer D (10 mM Tris-HCl (ph 7.6), 10 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10%
glycerol). The pellet was resuspended in an equal volume of Buffer C
(Buffer D plus 20% glycerol) and passed through a 21-gauge needle and
frozen several times on liquid N2 to lyse nuclei. After
centrifugation to remove debris, the supernatant was mixed with an
equal volume of Buffer D (nuclear fraction). Polysomes were prepared as
previously described (36). Polysome extract was pre-treated at 25 °C
for 15 min with ribonuclease A (37) when indicated, and the RNase was
inactivated with the ribonuclease inhibitor RNasin (Promega), prior
incubation of the extract with the labeled RNA. The amount of RNase A
used was determined by titration.
70 °C. For competition experiments, the extract was incubated simultaneously with the indicated amounts of unlabeled and labeled RNAs. All homoribopolymers (poly(A), poly(C), poly(G), and poly(U)) were from Sigma.
-32P]dCTP
(PerkinElmer Life Sciences) by PCR as in Ref. 39. Densitometry was done
using 1D Image Analysis Software (Kodak Digital Science).
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
Half-life determinations of
cat mRNA fused to complete mucin SMUG-L 3'-UTR and
deletion mutants. A, schematic representations of
complete SMUG-L and 3'-UTR deletion mutants are shown. All constructs
were done by PCR as described under "Experimental Procedures" using
PCR primers with restriction endonuclease sites (B,
BamHI; S, SmaI; H,
HindIII; E, EcoRI; X,
XhoI). The 5' and 3' intergenic regions (IR)
contain the original trans-splicing site (ag) and
polypyrimidine tract (pPy) for efficient mRNA
processing. Epimastigote forms of the parasite were transfected with
the indicated DNA constructs cloned in pTEX vector (33). B,
epimastigotes transfected with the recombinant DNAs described in
A were treated with 10 µg/ml ActD and total RNA was
prepared at the indicated times (0, 60, 120, and 180 min). Equal
amounts of RNA were analyzed by Northern blot. The same filter was
sequentially hybridized with cat, neo, and rRNA
probes. The hybridization performed with the neo probe
serves as an internal control of the experiment since this gene is
expressed from the same vector. C, quantitation of the bands
from the Northern blots shown in B. The half-life of each
transcript is indicated below the graphic. D, epimastigotes
transfected with SMUG-L and SMUG-L GRE constructions were treated
with 10 µg/ml ActD and total RNA was prepared at the indicated times
(0, 15, 30, 45, and 60 min). E, quantitation of the bands
from the Northern blots shown in D. In panels
C and E, the data were expressed as the mean
relative amount of mRNA ± the standard error of the media
(n = 3) at each time point after correction for the
level of rRNA. Differences between SMUG-L and each deletion mutant were
significant when comparing the means by Student's t test
(*, p < 0.05; **, p < 0.01).
GRE transcript had a shorter half-life
(t1/2 = 30 min), that is about 42% of that from
SMUG-L clone. GRE sequence is a G-rich element that contains the first
27 nt of the 3'-UTR downstream the stop codon and is composed by two
contiguous CGGGG pentamers (see below). Transcripts from two other
constructs, SMUG-L
2 and SMUG-L
3, had similar half-lives to those
of SMUG-L (t1/2 = 75 min and t1/2 = 65 min, respectively). Finally, SMUG-L
1 and SMUG-L
Sire deletion
mutants were transcribed into RNAs having increased half-lives
(t1/2 = 140 min) (Fig. 1, B and
C). Since the short interspersed repeat element (SIRE)
retrotransposon (40) is a large element (450 base pairs), some partial
deletion would be required to better define the region causing this
effect. Since in the half-life determination of clone SMUG-L
GRE
(Fig. 1B), less than 50% of the mRNA levels remained at
the first sampling time (60 min), the experiment was repeated taking
samples between 0 and 60 min. Thus, the half-life of SMUG-L
GRE was
better calculated and shown to be 30 min, identical to that indicated
in Fig. 1C (Fig. 1, D and E). These
results suggest that the sequences in the 3'-UTR could be divided into
several functional regions: 1) a positive G-rich element named GRE; 2)
one negative element between nucleotides 28 and 62 downstream stop
codon, named E1 for element 1; and 3) an AU-rich element between
nucleotides 272 and 318 involved in selective mRNA destabilization
in a stage-specific manner (see next section). The 3'-UTRs of SMUG-L
and SMUG-L
GRE were modeled using the Genequest program (Lasergene
Package, DNAstar Inc.) to predict if the deletion of GRE sequence would
have an effect on the three-dimensional structure of the RNA. Both
transcripts were found to share the same modeled structure, including
all loops of the 3'-UTR (data not shown). Thus, it is likely that the
sequence of the G-rich element is the one that confers the effect
reflected on mRNA stability, and not any modification of the whole
3'-UTR of the RNA molecule.
GRE was similar (117% of SMUG-L) than the
one obtained from parasites transfected with the complete contruct
SMUG-L, suggesting that this G-rich element does not modulate
translation efficiency. Conversely, SMUG-L
1 deletion mutant, whose
transcript has a larger half-life, also presented an increase in
translation (185% of SMUG-L). This suggests that SMUG-L
1 regulates
both mRNA stability and translation efficiency in a negative
manner. Moreover, SMUG-L
2 and SMUG-L
3 did not show a considerable
effect on translational activity (88% and 112% of SMUG-L,
respectively) (Fig. 2). Finally, the retrotransposon SIRE seems to
produce a positive effect on translation, since its deletion causes a
decrease in the ratio CAT activity/cat mRNA (15% of
SMUG-L construct). This result is interesting, because it was suggested
that SIRE exhibits another function in the process of mRNA
maturation (see "Discussion"). Sites for 5' end
trans-splicing and 3' end polyadenylation were the same in
all the mRNAs derived from the constructs made, as indicated under
"Experimental Procedures."
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Fig. 2.
Negative and positive
cis-elements within the 3'-UTR of SMUG-L mucin
mRNA modulate translation efficiency. A, mRNA
steady-state levels of epimastigote stage of the parasite transfected
with the DNA constructs shown in Fig. 1. The same filter was
sequentially hybridized with cat and rRNA probes.
B, thin layer chromatography of CAT activity assayed in the
same parasite population used in the experiment shown in A. C, bar representation of the ratio of CAT activity
(normalized to parasite number) versus cat
mRNA level (normalized to rRNA signal) is shown. Results are
expressed as the mean value ± the standard error media
(n = 3). Differences between SMUG-L, SMUG-L 1, and
SMUG-L
Sire were significant (*, p < 0.05, when
comparing the means by Student's t test).
GRE construct)
on mRNA stability was analyzed in different parasite stages and the results compared with those obtained with the constructs SMUG-L (complete 3'-UTR) and SMUG-L
AU (lacking the 44-nt AU-rich
instability element) (Fig.
3A). Epimastigote forms were
differentiated into the infective form of the parasite, metacyclic
trypomastigotes, and incubated with ActD to determine half-lives of the
transcripts (Fig. 3). The probe used in the Northern blot analysis
corresponds to the cat open reading frame. In the
epimastigote stage, both SMUG-L and SMUG-L
AU transcripts bearing the
GRE sequence within its 3'-UTR have similar half-lives
(t1/2 = 70 and t1/2 = 68 min,
respectively). On the other hand, transcripts from SMUG-L
GRE are
less stable (t1/2 = 30 min) (Fig. 3C). It
might be concluded that: 1) the GRE sequence in the epimastigote stage is involved in a selective mRNA stabilization process, and 2) the
ARE sequence seems not to be involved in mRNA stabilization in this
parasite form, since transcripts from both SMUG-L and SMUG-L
AU
constructs have similar half-lives (Fig. 3, B and
C) (see "Discussion").
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Fig. 3.
A novel GRE localized in the 3'-UTR of SMUG-L
confers mRNA stability in a stage-specific manner and is
functionally different to the AU-rich element. A,
schematic representation of SMUG-L (complete construct) and
SMUG-L GRE and SMUG-L
AU deletion mutants used to transfect
epimastigote stage of the parasite. The sequence that was deleted in
clone SMUG-L
GRE and SMUG-L
AU is indicated in the SMUG-L scheme.
B, Northern blot of total RNA from epimastigotes transfected
with the constructs shown in A. Epimastigotes were treated
with 10 µg/ml ActD, and total RNA was prepared at the indicated times
(0, 45, 60, 90, and 120 min). The same filter was sequentially
hybridized with cat, neo, and rRNA probes.
C, quantitation of cat mRNA levels from the
Northern blot shown in B. The data were expressed as the
mean relative amount of mRNA ± the standard error of the
media (n = 3) at each time point after correction for
the level of rRNA. Differences between SMUG-L
GRE and SMUG-L and
between SMUG-L
GRE and SMUG-L
AU were significant (*,
p < 0.05; **, p < 0.01 when comparing
the means by Student's t test). D,
epimastigote-derived metacyclic trypomastigotes were treated as
indicated in B. E, quantitation of cat
mRNA levels from the Northern blot shown in D. The data
were expressed as the mean relative amount of mRNA ± the
standard error of the media (n = 2) at each time point
after correction for the level of rRNA. Differences between SMUG-L
AU
and SMUG-L and between SMUG-L
AU and SMUG-L
GRE were significant
(**, p < 0.01, when comparing the means by Student's
t test). In panels C and E,
the half-life of each transcript is indicated below the
graphic.
GRE RNAs were
extremely short-lived (t1/2 < 10 min) as compared
with those from SMUG-L
AU, which have a t1/2 > 30 min (Fig. 3E). Thus, the instability of SMUG-L and
SMUG-L
GRE transcripts in the metacyclic trypomastigotes stage could
be due to the presence of the ARE sequence within its 3'-UTR.
Additionally, the same filter used to detect cat transcripts
was hybridized with a neo probe. Since the neomycin gene is flanked by
glyceraldehyde-3-phosphate dehydrogenase intergenic regions (33) in the
same plasmid bearing the cat reporter, it serves as an
internal control of half-life determinations. As seen in Fig. 3
(B and D), neomycin half-lives are similar in each parasite stage independently of the construct tested.
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Fig. 4.
GRE cis-element is bound by
specific RNA-binding proteins having different molecular masses.
A, the 32P-labeled in vitro
transcribed SMUG- L-GRE RNA was incubated with
total protein extract from the four life cycle parasite
stages: E, epimastigotes; Mt,
metacyclic trypomastigotes; T, cell-derived
trypomastigotes; and A, intracellular amastigotes. For the
analysis of RNA-protein interactions, the in vitro binding
reaction was electrophoresed on a 7% native polyacrylamide gel.
RNA-protein complexes (G-rich complexes) are indicated by
arrows 1, 2, and 3 on the
left side of the panel. B, SMUG-L-GRE
RNA was incubated with total protein extract from the epimastigote
stage of the parasite and an EMSA was performed as in A. The
localized RNA-protein complexes 1, 2, and 3 were cross-linked by UV
light irradiation and electrophoresed on 10% SDS-PAGE. No radiolabeled
proteins were seen when the lysate was pretreated with proteinase K or
when RNase was added before UV irradiation (data not shown). Molecular
size protein standards (kDa) are shown on the left side of
the figure. C, native EMSA of an epimastigote total protein
extract incubated with 32P-labeled SMUG-L-GRE
RNA and competed with increasing amounts of unlabeled homoribopolymers
(poly(A), -(C), -(G), and -(U)) or without competitor ( );
RNA, probe alone.
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Fig. 5.
Identification of the minimal
cis-element required for GRE binding and subcellular
localization of GRE RNA-binding proteins. A, sequence
of the RNA oligonucleotides used in B: GRE,
complete G-rich element; GRE-1 and GRE-2, first-
and second halves of GRE element, respectively. B, epimastigote forms
of the parasite were lysed with Nonidet P-40 as described under
"Experimental Procedures" (T) or separated into
fractions containing nuclei (N) and cytosol (C).
Materials obtained from equal number of parasites (10 × 106) were utilized for an in vitro binding
reaction and electrophoresed in a native polyacrylamide gel with the
indicated RNA probes. The position of the three G-rich complexes (1, 2 and 3) is indicated with arrows on the left side of the
panel. A small arrow with an asterisk denotes the
position of an artifact band, due to the RNA probe.
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Fig. 6.
ARE RNA-binding proteins are developmentally
regulated and have different molecular masses in the parasite life
cycle stages. A, the 32P-labeled in
vitro transcribed SMUG-L-AU RNA was incubated with
total protein extract from the four life cycle parasite stages:
E, epimastigotes; Mt, metacyclic trypomastigotes;
T, cell-derived trypomastigotes; and A,
intracellular amastigotes. For the analysis of RNA-protein
interactions, the in vitro binding reaction was
electrophoresed on a 7% native polyacrylamide gel. The
arrows indicate the position of the different
ribonucleoprotein complexes formed. E-ARE-BP denotes the
position of an specific epimastigote AU-rich RNA-binding protein.
ARE-BPs, AU-rich element RNA-binding proteins present in the
other three parasite stages. B, SMUG-L-AU RNA
was incubated with total protein extract from the four life cycle
parasite stages and an EMSA was performed as in A. The
localized RNA-protein complexes were cross-linked by UV light
irradiation and further electrophoresed on 10% SDS-PAGE. No
radiolabeled proteins were seen when the lysate was pretreated with
proteinase K or when RNase was added before UV irradiation (data not
shown). Molecular size protein standards (kDa) are shown on the
left side of the figure.
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Fig. 7.
. Specificity of E-ARE-BP and T-ARE-BPs.
A, native EMSA of an epimastigote total protein extract
incubated with the 32P-labeled SMUG-L-AU probe
and competed with homoribopolymers (poly(A), -(C), -(G), and -(U)) or
without competitor ( ); RNA, probe alone. B,
native EMSA of epimastigote total extract binding reaction competed
with in vitro transcribed poly(U), SMUG-L-AU
antisense, and SMUG-L-AU sense RNAs. The position of the
E-ARE-BP is indicated on the left side of the panel. The
molar excess of each competitor is indicated above each
panel. C, native EMSA of a trypomastigote total protein
extract incubated with the 32P-labeled
SMUG-L-AU probe and competed with a 1000-fold molar excess
of homoribopolymers (poly(A), -(C), -(G), and -(U)) or without
competitor (
); RNA, probe alone. D, native EMSA
of a trypomastigote total extract binding reaction competed with
in vitro transcribed SMUG-L-AU antisense and
SMUG-L-AU sense RNAs. The position of T-ARE-BPs is
indicated on the left side of the panel. The molar excess of
each competitor is indicated above each panel.
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Fig. 8.
E-ARE-BP recognized the AU-rich RNA
instability element in the cytoplasm, while T-ARE-BPs are localized in
nucleus and cytoplasm. A, subcellular localization of
the E-ARE-BP RNA-binding protein determined by in vitro
binding reaction of different protein extracts with the
SMUG-L-AU RNA. Epimastigote stage of the parasite were
lysed with Nonidet P-40 as described under "Experimental
Procedures" (T) or separated into fractions containing
nuclei (N), cytosol (C), polysomes
(P), and postribosomal supernatant (PS). Equal
number of parasite (10 × 106) equivalents were
utilized for the in vitro binding reactions and
electrophoresed in a native polyacrylamide gel with the indicated RNA
probes. B, polysome extract of the epimastigote stage of the
parasite was pre-treated (+) or not ( ) with RNase A, and inactivated
with RnasiN as indicated under "Experimental Procedures," prior to
incubation with the SMUG-L-AU probe. C,
subcellular localization of trypomastigote ARE-BPs
(T-AREBPs) determined as in panel
A.
and some cytokine and proto-oncogene mRNAs
(44). A polysome fraction (P) of T. cruzi
epimastigotes was prepared as described previously (36) in the presence
of cycloheximide to freeze ribosomes. After extract preparation and centrifugation through a sucrose cushion, the supernatant was saved as
a postribosomal preparation (PS) and the pellet was saved as
polysomes (P). All the extracts were analyzed in an in
vitro binding reaction with the SMUG-L-AU RNA
template. The polysome extract was shown to have some AU-rich sequence
binding activity, but minimal in comparison to the one observed in the
postribosomal fraction (Fig. 8A). To further determine if
the lack of a strong shifted band in the polysome fraction was due to
the presence of some endogenous U-rich RNA competitor that might be
sequestering part of E-ARE-BP, the extract was pre-treated with
ribonuclease A (RNase A) as described previously (37), and the nuclease
was inactivated prior to perform the in vitro binding
reaction with the SMUG-L-AU RNA probe. The result shown in
Fig. 8B demonstrate that, in the presence of RNase, the
binding of E-ARE-BP is increased 4.5-fold, suggesting that there might
be some RNA competing with the labeled AU-rich RNA in the polysome
fraction. Moreover, the RNA probe remains intact after incubation with
the polysome extract. Thus, the absence of such a strong band in these
fraction was not due to the presence of a polysome-associated nuclease
that could recognize the ARE sequence (Fig. 8B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,
named here E1 and localized between nucleotides 28 and 62 of the
3'-UTR, increases the half-life of a cat reporter mRNA
(Fig. 1), suggesting that this sequence acts as a negative element.
Finally, deletion of the 450-base pair retrotransposon SIRE also
produces the same effect as the element E1, but, given the large size
of SIRE sequence, further work is required to confirm this effect. It
was shown previously that SIRE is responsible for the down-regulation
of gene expression of the TCP2
ribosomal protein by altering its trans-splicing efficiency (45). Thus, different functions
might be assigned to sequences within this retrotransposon. Indeed, it
was reported that U-rich regions and also the length of the 3'-UTR
positively regulate mRNA polyadenylation and the translation efficiency of a reporter gene (11). Although GRE sequence is sufficient
to up-regulate SMUG mRNA abundance, E1 has a dual effect on
mRNA stability and translation, regulating both processes in a
negative manner. It is not unprecedented for a single element to have
two functions, since AU-rich sequences within the 3'-UTR of TNF-
affect both mRNA abundance and translation efficiency (46-48).
AU
constructs have similar half-lives in the latter stage (Fig. 3). These
results further support the idea that the RNA-binding protein(s) that recognize the ARE in the epimastigote stage of the parasite, might provide resistance to endo- or exonucleolytic cleavage rather than
providing actively mRNA protection. Conversely, GRE sequences have
a different effect on mRNA stability throughout parasite development. It up-regulates SMUG mRNA abundance in the
epimastigote stage, since deletion of the GRE motif makes the mRNA
more labile (Fig. 3, B and C). The presence of
the ARE sequence within the 3'-UTR of mucin SMUG mRNA also have
been shown to modulate translation efficiency in a positive manner (1).
In contrast, GRE had no considerable effect on translational levels,
suggesting that both elements might coordinately cooperate in the
in vivo regulation of SMUG mRNA abundance in the
epimastigote stage of the parasite, but not in translation. Coordinated
interaction between different negative and positive
cis-elements was observed in the 3'-UTR of procyclic
mRNAs of African trypanosomes, affecting both mRNA abundance
and translation efficiency (12).
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Fig. 9.
A model for the post-transcriptional
regulatory mechanism acting on mucin SMUG mRNA stability in
different stages of T. cruzi development.
GRE RNA-binding proteins (G-80, G-35,
G-50, and G-66) are present in both epimastigote
and metacyclic trypomastigote stages of the parasite. E-ARE-BP (a
100-kDa ARE-binding protein) is only detected in the epimastigote stage
and might protect mucin SMUG mRNA from degradation. The 45-50-kDa
T-ARE-BPs are only detected in the other three developmental stages of
the parasite. Additional cellular factors forming part of an mRNA
decay machinery or translational apparatus might interact with the
T-ARE-BPs and E-ARE-BP, respectively.
A model for the post-transcriptional regulatory mechanism acting on
mucin SMUG mRNA and mediated by ARE and GRE RNA-binding proteins is
shown in Fig. 9. E-ARE-BP, only expressed in the epimastigote stage,
might be a positive trans-acting factor interacting with the
ARE and protecting SMUG mRNA from degradation. E-ARE-BP binding could also prevent the association of the destabilizing factor(s) to
those mRNAs, possibly through competition for binding to similar cis-elements. Indeed, E-ARE-BP might be one of the proteins
involved in the modulation of the translation activity mediated by the ARE motif (1), probably through the interaction with other cellular
factors of the translational apparatus. On the other hand, GRE
RNA-binding proteins are always present during the life cycle of
T. cruzi (Fig. 9). The possibility that an ARE-GRE-complex exists in vivo, and that this whole complex or some
complex-forming proteins interact with a poly(A)binding protein or
other cellular factor(s) to prevent the attack of a deadenylase
activity, remains to be investigated. It is well known that, in
mammalian cells, a large complex is formed by several proteins having
different affinities for poly(C) homoribopolymer, such as the assembly
of the -globin mRNA stability complex in the pyrimidine-rich
region of the globin 3'-UTR (22).
The results obtained by subcellular fractionation suggest that E-ARE-BP is localized in the cytoplasm or only recognized the RNA in this cellular compartment, where mRNA decay or translational processes take place. Future experiments on Western blot analysis would permit us to determine if E-ARE-BP is also present in the nucleus and, thus, is recruited by some complex-forming proteins. Conversely, the ARE-BPs, at least in the trypomastigote stage, are present in similar amounts in both nucleus and cytoplasm and might be shuttling RNA-binding proteins (Fig. 8). G-complexes forming proteins, at least G-complex 2, might be formed by RNA-binding factors that showed a shuttling behavior between nucleus and cytoplasm. Consequently, it is possible that those GRE RNA-binding proteins might protect the messenger during transport between both compartments. Several proteins in higher eukaryotes were shown to present a shuttling behavior between nucleus and cytoplasm (18, 20, 49). In trypanosomes a classical nuclear localization signal was identified and shown to be functional in the La and histone H2B proteins (50). A regulated nuclear-cytoplasm export pathway mediated by CRM1 also might be present in kinetoplastid parasites, since leptomycin B affects the axenic growth of the epimastigote form of the parasite.2 Leptomycin B inhibits the formation of the complex formed by nuclear export signal-containing proteins, RanGTP, and the receptor CRM1 (51).
Post-transcriptional regulatory mechanisms, such as the ones mediated
by ARE or GRE sequences, may be required for a quick response to change
mucin core molecules expression pattern, triggering parasite adaptation
to sudden changes on the environment. In this regard, expression of the
correct surface mucin coat may be of central importance for parasite
survival. Identification of an in vivo role for these ARE
and GRE RNA-binding proteins in the mRNA stability of T. cruzi transcripts may allow proposal of a model of RNA metabolism
and maturation in parasites that are deficient in the regulation by RNA
polymerase II transcription.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to Berta Franke de Cazzulo and Liliana Sferco for parasite cultures. We are also most grateful to Dr. J. M. Kelly for providing us the pTEX vector, and Drs. J. J. Cazzulo and S. Silberstein for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Agencia Nacional de Promoción Científica y Tecnológica (Argentina) and the United Nations Developmental Program/World Bank/World Health Organization Special Program for Tropical Diseases.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.
Postgraduate fellow of the Consejo Nacional de Investigaciones
Científicas y Técnicas, Argentina.
§ Supported in part by an International Research Scholar grant from the Howard Hughes Medical Institute. Researcher of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina. To whom correspondence should be addressed: Inst. de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín, INTI, Av. Gral. Paz s/n, Edificio 24, Casilla de Correo 30, 1650 San Martín, Pcia. de Buenos Aires, Argentina. Tel.: 54-11-4752-9639; Fax: 54-11-4580-7255; E-mail: cfrasch@iib.unsam.edu.ar.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M010959200
2 I. D'Orso and A. C. C. Frasch, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: UTR, untranslated region; ARE, AU-rich element; GRE, G-rich element; ARE-BP, AU-rich element binding protein; ActD, actinomycin D; EMSA, electrophoresis mobility shift assay; CAT, chloramphenicol acetyltransferase; nt, nucleotide(s); SIRE, short interspersed repeat element; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PCR, polymerase chain reaction; TBE, Tris-borate EDTA; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Di Noia, J. M.,
D'Orso, I.,
Sanchez, D. O.,
and Frasch, A. C.
(2000)
J. Biol. Chem.
275,
10218-10227 |
2. | Pays, E., and Nolan, D. P. (1998) Mol. Biochem. Parasitol. 91, 3-36[CrossRef][Medline] [Order article via Infotrieve] |
3. | Roditi, I., Furger, A., Ruepp, S., Schurch, N., and Butikofer, P. (1998) Mol. Biochem. Parasitol. 91, 117-130[CrossRef][Medline] [Order article via Infotrieve] |
4. | Cross, G. A., Wirtz, L. E., and Navarro, M. (1998) Mol. Biochem. Parasitol. 91, 77-91[CrossRef][Medline] [Order article via Infotrieve] |
5. | Ben Amar, M. F., Jefferies, D., Pays, A., Bakalara, N., Kendall, G., and Pays, E. (1991) Nucleic Acids Res. 19, 5857-5862[Abstract] |
6. | Lee, M. G. (1996) Mol. Cell. Biol. 16, 1220-1230[Abstract] |
7. | Matthews, K. R., Tschudi, C., and Ullu, E. (1994) Genes Dev. 8, 491-501[Abstract] |
8. | Vanhamme, L., and Pays, E. (1995) Microbiol. Rev. 59, 223-240[Abstract] |
9. |
Wilson, M. E.,
Paetz, K. E.,
Ramamoorthy, R.,
and Donelson, J. E.
(1993)
J. Biol. Chem.
268,
15731-15736 |
10. | Berberof, M., Vanhamme, L., Tebabi, P., Pays, A., Jefferies, D., Welburn, S., and Pays, E. (1995) EMBO J. 14, 2925-2934[Abstract] |
11. | Nozaki, T., and Cross, G. A. (1995) Mol. Biochem. Parasitol. 75, 55-67[CrossRef][Medline] [Order article via Infotrieve] |
12. | Furger, A., Schurch, N., Kurath, U., and Roditi, I. (1997) Mol. Cell. Biol. 17, 4372-4380[Abstract] |
13. |
Hotz, H. R.,
Hartmann, C.,
Huober, K.,
Hug, M.,
and Clayton, C.
(1997)
Nucleic Acids Res.
25,
3017-26 |
14. |
Coughlin, B. C.,
Teixeira, S. M.,
Kirchhoff, L. V.,
and Donelson, J. E.
(2000)
J. Biol. Chem.
275,
12051-12060 |
15. | Caput, D., Beutler, B., Hartog, K., Thayer, R., Brown-Shimer, S., and Cerami, A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1670-1674[Abstract] |
16. | Shaw, G., and Kamen, R. (1986) Cell 46, 659-667[Medline] [Order article via Infotrieve] |
17. | Chen, C. A., and Shyu, A. (1995) Trends Biochem. Sci. 20, 465-470[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Fan, X. C.,
and Steitz, J. A.
(1998)
EMBO J.
17,
3448-3460 |
19. |
Peng, S. S.,
Chen, C. Y.,
Xu, N.,
and Shyu, A. B.
(1998)
EMBO J.
17,
3461-3470 |
20. |
Loflin, P.,
Chen, C. Y.,
and Shyu, A. B.
(1999)
Genes Dev.
13,
1884-1897 |
21. | Wang, X., Kiledjian, M., Weiss, I. M., and Liebhaber, S. A. (1995) Mol. Cell. Biol. 15, 1769-1777[Abstract] |
22. |
Wang, Z.,
Day, N.,
Trifillis, P.,
and Kiledjian, M.
(1999)
Mol. Cell. Biol.
19,
4552-4560 |
23. | Kiledjian, M., DeMaria, C. T., Brewer, G., and Novick, K. (1997) Mol. Cell. Biol. 17, 4870-4876[Abstract] |
24. |
Pereira-Chioccola, V. L.,
Acosta-Serrano, A.,
Correia de Almeida, I.,
Ferguson, M. A.,
Souto-Padron, T.,
Rodrigues, M. M.,
Travassos, L. R.,
and Schenkman, S.
(2000)
J. Cell Sci.
113,
1299-1307 |
25. |
Van Klinken, B. J. W.,
Dekker, J.,
Buller, H. A.,
and Einerhand, A. W. C.
(1995)
Am. J. Physiol.
269,
G613-G627 |
26. |
Di Noia, J. M.,
D'Orso, I.,
Aslund, L.,
Sanchez, D. O.,
and Frasch, A. C.
(1998)
J. Biol. Chem.
273,
10843-10850 |
27. | Frasch, A. C. (2000) Parasitol. Today 16, 282-286[CrossRef][Medline] [Order article via Infotrieve] |
28. | Zingales, B., Pereira, M. E., Oliveira, R. P., Almeida, K. A., Umezawa, E. S., Souto, R. P., Vargas, N., Cano, M. I., da Silveira, J. F., Nehme, N. S., Morel, C. M., Brener, Z., and Macedo, A. (1997) Acta Trop. 68, 159-173[CrossRef][Medline] [Order article via Infotrieve] |
29. | Gonzalez Cappa, S. M., Bijovsky, A. T., Freilij, H., Muller, L., and Katzin, A. M. (1981) Medicina 41, 119-120[Medline] [Order article via Infotrieve] |
30. |
Charest, H.,
Zhang, W. W.,
and Matlashewski, G.
(1996)
J. Biol. Chem.
271,
17081-17090 |
31. | Maranon, C., Puerta, C., Alonso, C., and Lopez, M. C. (1998) Mol Biochem. Parasitol 92, 313-324[CrossRef][Medline] [Order article via Infotrieve] |
32. | Martinez-Calvillo, S., Lopez, I., and Hernandez, R. (1997) Gene (Amst.) 199, 71-76[CrossRef][Medline] [Order article via Infotrieve] |
33. | Kelly, J. M., Ward, H. M., Miles, M. A., and Kendall, G. (1992) Nucleic Acids Res. 20, 3963-3969[Abstract] |
34. | Smith, C. (1998) in RNA-Protein Interactions: A Practical Approach (Hames, B., ed) , pp. 109-136, Oxford University Press, Inc., New York |
35. |
Mahmood, R.,
and Ray, D. S.
(1998)
J. Biol. Chem.
273,
23729-23734 |
36. | Gonzalez, N., and Cazzulo, J. (1989) Biochem. Pharmacol. 38, 2873-2877[Medline] [Order article via Infotrieve] |
37. | Akhtar, A., Zink, D., and Becker, P. B. (2000) Nature 407, 405-409[CrossRef][Medline] [Order article via Infotrieve] |
38. | Fourney, R. M., Miyakoshi, J., Day, R. S., III, and Paterson, M. (1978) Focus 10, 5-9 |
39. | Mertz, L., and Rashtchian, A. (1994) Focus 16, 45-48 |
40. | Vazquez, M. P., Schijman, A. G., and Levin, M. J. (1994) Mol. Biochem. Parasitol. 64, 327-336[CrossRef][Medline] [Order article via Infotrieve] |
41. | Winstall, E., Gamache, M., and Raymond, V. (1995) Mol. Cell. Biol. 15, 3796-3804[Abstract] |
42. |
Nagy, E.,
and Rigby, W. F.
(1995)
J. Biol. Chem.
270,
2755-2763 |
43. | Zhang, W., Wagner, B. J., Ehrenman, K., Schaefer, A. W., DeMaria, C. T., Crater, D., DeHaven, K., Long, L., and Brewer, G. (1993) Mol. Cell. Biol. 13, 7652-7665[Abstract] |
44. | Kruys, V., Marinx, O., Shaw, G., Deschamps, J., and Huez, G. (1989) Science 245, 852-855[Medline] [Order article via Infotrieve] |
45. | Vazquez, M. P., and Levin, M. J. (1998) Mem. Inst. Oswaldo Cruz 93, 161[Medline] [Order article via Infotrieve] |
46. | Xu, N., Chen, C. Y., and Shyu, A. B. (1997) Mol. Cell. Biol. 17, 4611-4621[Abstract] |
47. |
Lai, W. S.,
Carballo, E.,
Strum, J. R.,
Kennington, E. A.,
Phillips, R. S.,
and Blackshear, P. J.
(1999)
Mol. Cell. Biol.
19,
4311-4323 |
48. |
Carballo, E.,
Lai, W. S.,
and Blackshear, P. J.
(1998)
Science
281,
1001-1005 |
49. | Shyu, A. B., and Wilkinson, M. F. (2000) Cell 102, 135-138[Medline] [Order article via Infotrieve] |
50. |
Marchetti, M. A.,
Tschudi, C.,
Kwon, H.,
Wolin, S. L.,
and Ullu, E.
(2000)
J. Cell Sci.
113,
899-906 |
51. | Fornerod, M., Ohno, M., Yoshida, M., and Mattaj, I. W. (1997) Cell 90, 1051-1060[Medline] [Order article via Infotrieve] |