(Received for publication, September 20, 1996, and in revised form, November 22, 1996)
From the Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, 28049 Madrid, Spain
The genomic organization and expression of the
hsp70 genes of Leishmania infantum were
examined. In the cluster there are at least six copies of the
hsp70 genes arranged in a head-to-tail tandem of
3.8-kilobase repetition units. The hsp70 gene copy (gene 6)
located at the 3 end of the tandem has a 3
-untranslated region highly
divergent in sequence relative to the 3
-untranslated region of the
rest of hsp70 gene copies (genes 1-5). Nuclease S1
protection assays indicated that the steady-state level of the
mRNAs derived from gene 6 is about 50-fold more abundant than the
transcript level derived from genes 1-5. Nuclear run-on assays showed,
however, that all hsp70 genes are transcribed at similar
rates. Thus, it is likely that the differences in the steady-state
levels of the transcripts from the hsp70 genes should be
associated with variations in their processing or maturation rates.
While the abundance of the mRNAs derived from hsp70
genes 1-5 is increased by heat shock, the hsp70 gene 6 mRNA level remains unaffected. Our data showed that ongoing protein
synthesis is required for the maintenance of the heat inducement,
depicting, thus, a post-transcriptional mechanism of positive
regulation involving a labile protein factor that would be either
induced or activated during heat shock.
Leishmania parasites experience a shift in temperature during their life cycle while being transferred from the sandflies, as flagellated promastigotes, to the vertebrate host where they enter into macrophages and transform to aflagellated amastigotes. This change in temperature is known to affect gene expression as well as stage differentiation (1). Although most of the eukaryotes respond to a heat shock by increasing the rate of transcription of specific genes to attain high levels of the heat-shock proteins (hsp),1 Leishmania and other trypanosomes do not induce the transcription of their hsp genes during a heat stress. Instead, the levels of hsp appear to be post-transcriptionally regulated. In fact, it has been shown by analysis of the expression of the hsp70 and hsp83 genes of Leishmania major and Leishmania donovani that there is not transcriptional activation of these genes when the parasites are exposed to a heat shock (2). Similarly, although no transcriptional activation of the Leishmania amazonensis hsp83 genes is induced upon a heat shock, hsp83 transcripts accumulate in this condition (3). The accumulation of the hsp83 transcripts results mainly from differences in stability since while the hsp83 mRNAs are rapidly degraded at the normal temperature they becomes stable at 35 °C (3). The regulation of Trypanosoma brucei hsp70 transcripts (4, 5) and the control of other stage-regulated genes seems to be also post-transcriptional (6-14).
Post-transcriptional regulation is probably a consequence of the
clustering as tandem repeats of most Trypanosomatid genes and of the
transcription of the genes from the cluster as polycistrons co-transcriptionally processed by both 5 trans-splicing of
a capped leader RNA and polyadenylation (15). Therefore, regulation of
the expression of individual genes within the cluster cannot occur at
the level of transcription initiation. Particularly, the genes of the
hsp70 and hsp83 families have been chosen as models for the study of
the organization and expression of trypanosome genes. Hsp70-encoding
genes have been found repeated and tandemly organized in T. brucei (16), Trypanosoma cruzi (17), L. major (18, 19), and L. amazonensis (20). Similarly,
genes coding for hsp83 proteins have been found repeated and tandemly
organized in T. cruzi (21), T. brucei (22), and
L. mexicana amazonensis (23). In L. major, in
addition to the four tandemly clustered hsp70 genes, a fifth
hsp70 gene is located in a separate locus (18).
Interestingly, while the expression levels of the tandemly linked
hsp70 genes are increased after a heat-shock treatment at
37 °C the nonlinked gene is unaffected by a temperature shift (18).
In the present report we present the analysis of the regulation of the
expression of the hsp70 genes from Leishmania
infantum. As a first step in this study we examined the genomic
organization of these genes. We found that the L. infantum
hsp70 genes are located in a single cluster formed by six
hsp70 units in a head-to-tail tandem array. We observed that
when the parasites are grown at 26 °C the abundance of the
steady-state transcripts derived from the gene located at the 3 end of
the cluster (gene 6) accumulates to higher levels than the transcripts
derived from the rest of hsp70 genes (genes 1-5). Also, we
detected that when the parasites are grown at 37 °C the levels of
transcripts derived from the hsp70 genes 1-5 results
increased by the heat treatment in contrast to the transcripts level
from gene 6 that remains unaffected. Data from several
experimental approaches indicate that the differential regulation of
L. infantum hsp70 genes must be occurring at the post-transcriptional level by mechanisms involving specific sequences of the 3
-untranslated regions (UTR).
Promastigotes of L. infantum
(MHOM/FR/78/LEM 75) were cultured in vitro at 26 °C in
RPMI 1640 medium (Life Technologies, Inc., Paisley, UK) supplemented
with 10% heat-inactivated fetal calf serum (Flow Laboratories, UK). In
all the experiments logarithmic phase cultures (5-9 × 106 promastigotes ml1) were used.
Clone B2cDNA, a L. infantum
hsp70 cDNA isolated by immunological screening of a -gt11
cDNA expression library, has been previously described (24). This
cDNA was cloned into the EcoRI site of plasmid pUC18. A
330-bp DNA fragment, named B2 3
UTR-II, was obtained after
SalI digestion of clone pB2cDNA (Fig. 1A).
This fragment was cloned in the SalI site of pUC18 and the
resulting clone named pU3
UTR-II. It must be noted that this fragment
exclusively contains 3
-UTR sequences of an L. infantum
hsp70 gene (gene 6, see below).
The L. infantum EMBL-3 genomic library used in this work has
been previously described (25). Screening was carried out by standard
techniques (26) using the 32P-labeled B2cDNA as probe.
Seven positive clones, B2g1 to B2g7, were isolated. Similarly, a
screening was carried out on a L. infantum oligo(dT)-primed
lambda-gt11 cDNA expression library (27) using the B2 3UTR-II DNA
fragment as probe. From this screening two cDNA clones, 70IIA-1 and
70IIA-2, were isolated.
Probe 3UTR-I was obtained by PCR amplification from the 1.5-kb
SalI-BamHI fragment (clone B2g6; Fig.
1A), subcloned in pUC18, using the oligonucleotide 70-I
(5
-CACACCCAAGTACACGTCAG-3
) and the M13/pUC sequencing primer (
20)
17-mer (New England BioLabs, Inc.). Standard PCR conditions
(Perkin-Elmer) in the presence of 5% dimethyl sulfoxide were used. The
cycle profile, repeated 30 times, was 1 min at 94 °C, 1 min at
55 °C, and 1 min 72 °C. The PCR product was digested with
SalI, and the resulting 146-bp fragment was purified over a
spin bind column (FMC Bio Products) and cloned in a
SalI-SmaI digested pBlueScript SK(
) vector. The resulting clone was named pB3
UTR-I.
Clone pTc3 contains the
-tubulin gene of T. cruzi (28). Clone pRIB contains a 590-bp
SmaI-HindIII fragment of the L. infantum 24S
rRNA gene.2
DNA sequencing was conducted on double-stranded DNA by the dideoxy chain termination method (29), using the SequenaseTM kit (United States Biochemical Corp.). The nucleotide sequence of the cDNAs was determined in both strands using internal synthetic oligonucleotides.
The restriction map of the inserts of the B2g1 to B2g7 genomic clones
was determined for a variety of restriction enzymes (Fig.
1A). The consecutive 0.3-kb
EcoRI-BamHI, 2.1-kb
BamHI-SalI, and 1.5-kb
SalI-BamHI genomic fragments, conforming the 5
repetition unit of the L. infantum hsp70 gene cluster, were
subcloned in pBlueScript SK(
) vector (Stratagene). Also, the 5.72-kb
SalI fragment from clone B2g1, containing the 3
-UTR of the
hsp70 gene located at 3
end of the cluster (see Fig.
1A and text for details), was subcloned in pBlueScript. In
order to obtain the complete sequence of the 2.1-kb
BamHI-SalI fragment exonuclease III/mung bean
nuclease deletions were carried out as described elsewhere (24).
Synthetic oligonucleotides were used for sequencing of the other
fragments. The analysis of the nucleotide and amino acid sequences was
done using the University of Wisconsin Genetics Computer Group programs
(30) and by accessing the GenBank and EMBL data bases.
L. infantum
DNA and RNA were isolated as described previously (17, 31).
Promastigote total DNA was digested with a variety of restriction
endonucleases, electrophoresed in 0.8% agarose gels, and transferred
to nylon membranes (Hybond-N, Amersham Corp.) by standard methods (26).
Five µg per lane of total RNA was size separated on 1%
agarose-formaldehyde gels (32) and electrophoresed to nylon membranes
using a LKB system (Pharmacia Biotech Inc.). Hybridizations, either for
DNA or RNA analysis, were performed in 50% formamide, 6 × SSC,
0.1% SDS, and 0.25 mg ml1 herring sperm DNA at 42 °C
overnight. Final posthybridization washes were performed in 0.1 × SSC, 0.2% SDS at 50 °C for 1 h. For reuse, blots were treated
with 0.1% SDS for 30 min at 95 °C to remove the previously
hybridized probes. Removal of the probes was verified by
autoradiography.
Two oligonucleotides derived
from the divergent 3-UTR sequences were synthesized (Isogen): 70-I,
5
-CACACCCAAGTACACGTCAG-3
(reverse and complementary to nucleotides
2079-2099; Fig. 2A), and 70-II,
5
-GGGAAGCCCCACAGCGGAAAAGTGG-3
(reverse and complementary to
nucleotides 525-550; Fig. 2B). The oligonucleotides were
labeled with 50 µCi of [
-32P]ATP (6000 Ci/mmol;
Amersham Corp.) using the T4-polynucleotide kinase kit (Boehringer
Mannheim). The specific activity of the labeling was 109
cpm/µg. A molar excess (0.07 pmol) of 32P-labeled
oligonucleotide was hybridized with 2 µg of L. infantum poly(A+) RNA (purified by oligo(dT)-cellulose; Boehringer
Mannheim) in a 25-µl final volume containing 300 mM NaCl,
20 mM Tris-HCl, pH 8.0, and 1 mM EDTA. After
heating for 15 min at 75 °C, hybridization was performed for 3 h at 52 °C. In parallel, as controls, hybridizations of the
oligonucleotides to 4 µg of Escherichia coli rRNAs
(Boehringer Mannheim) were carried out. After ice-cooling of the
hybridization reaction, 25 µl of 2 × S1 buffer (66 mM NaAc, pH 4.5, 100 mM NaCl, and 0.06 mM ZnS04) and 40 units of nuclease S1
(Boehringer Mannheim) were added. After 15 min of incubation at
37 °C the reaction was stopped by adding an equal volume of loading
buffer (95% formamide, 10 mM EDTA, 0.1% bromphenol blue,
and 0.1% xylene cyanol). Finally, 6-µl samples were loaded on a 15%
polyacrylamide, 7 M urea sequencing gel for 4 h at 60 watts. After drying the gel was exposed to a x-ray film at
70 °C
for several hours. The autoradiographs were scanned with a laser
densitometer (Image Quant 2.0), and the relative densities of the bands
were determined.
Nuclear Run-on Assays
Promastigote cultures in logarithmic
phase growth (5 × 106 parasites ml1)
were preincubated at 37 °C during 0 (26 °C, control), 10, 30, and
60 min. At the indicated times, 10-ml aliquots were harvested, and the
parasites were suspended in 500 µl of ice-cold hypotonic buffer (0.25 M sucrose, 5 mM Hepes, pH 7.5, 1 mM
spermidine, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM EGTA, and 1 mM
dithiothreitol). Nonidet P-40 and Triton X-100 were added to a final
concentration of 0.5% each, and the cells were lysed by vigorously
vortexing for 30 s. Immediately, 2 volumes of ice-cold 2 × nuclei washing buffer (40 mM Tris-HCl, pH 7.5, 0.64 M sucrose, 1 mM spermidine, 0.1 mM
phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM
EGTA, 1 mM dithiothreitol, and 60 mM KCl) were
added and mixed by vortexing. The nuclei were pelleted (3000 × g), washed, and stored in 100 µl of nuclei storage buffer
(50% glycerol, 4 mM MnCl2, 1 mM
MgCl2, 0.1 mM EDTA, 50 mM Hepes, pH
7.5, and 5 mM dithiothreitol) at
70 °C, until use.
After thawing, 1 volume of 2 × transcription buffer (0.1 M Hepes, pH 7.5, 0.2 M KCl, 8 mM
dithiothreitol, 60 µM EDTA, 2 mM ATP, 1 mM CTP, 1 mM GTP, 17.6 mM creatine
phosphate, and 80 µg ml
1 creatine kinase (Boehringer
Mannheim)) was added. The run-on transcripts were labeled by adding 100 µCi of [
-32P]UTP (3000 Ci mmol
1)
(Amersham Corp.). Nuclei isolated from heat-shocked parasites were
labeled during 10 min at 37 °C, while those from control parasites
were labeled at 26 °C for the same time. The reaction was stopped by
addition of DNase I (RNase-free) and MgCl2 to final concentrations of 25 µg ml
1 and 5 mM,
respectively, and incubation for 20 min at 37 °C. Subsequently, the
reaction continued in the presence of 0.15 mg ml
1
proteinase K, 0.5% SDS, and 5 mM EDTA for 20 min at
37 °C. The radiolabeled nascent RNA was extracted by
phenol:chloroform. Non-incorporated isotopes were separated from the
labeled product on a Sephadex G-50 column.
A 5-µg sample of each plasmid to be tested was linearized, denatured,
and applied onto Zeta-probe membranes (Bio-Rad) in a vacuum slot-blot
apparatus according to the manufacturer's instructions. The membrane
was then subjected to hybridization with the purified labeled RNA
(2-6 × 106 cpm ml1) in a solution
containing 50% formamide, 6 × SSC, 0.1% SDS, and 0.25 mg
ml
1 herring sperm DNA for 3 days at 42 °C.
Subsequently, the filters were washed at room temperature for 15 min in
2 × SSC, followed by a wash at 65 °C for 30 min in 2 × SSC, and a final wash at 37 °C for 20 min in 2 × SSC, 10 µg
ml
1 of RNase A.
Genomic Organization of the L. infantum hsp70 Genes
Recently, we reported the isolation of a cDNA coding for a
fragment of the L. infantum hsp70 gene by immunoscreening of
a cDNA expression library (24). The identified cDNA clone,
named B2, contains the sequence coding for the carboxyl-terminal 20 amino acids and for 330 bp of the 3-UTR. In order to analyze the
genomic organization of the hsp70 genes, L. infantum DNA was digested with several restriction enzymes,
transferred onto nylon membranes, and probed with clone B2 (Fig.
1). The presence of a 3.8-kb hybridization band in the
lanes containing the DNA digested either with SalI or
BamHI was taken as an indication of the presence of several
copies of the hsp70 gene which are arranged in tandem and
that these enzymes cut only once within the repeat. In addition, the
fact that others restriction enzymes (HindIII and
EcoRI) produce a single hybridizing band supported the fact
that the hsp70 genes must be clustered in a single
chromosomal locus.
To analyze in more detail the genomic organization of the
hsp70 genes, a L. infantum genomic library was
screened with the B2cDNA probe. Seven clones, named B2g1 to B2g7,
were isolated and physically mapped (Fig. 1A). The
restriction analysis of those clones confirmed that the
hsp70 genes are tandemly arrayed. Southern blot analysis
indicated that probe B2cDNA hybridizes more strongly with clones
B2g1-5 and B2g7 than with clone B2g6 (data not shown), suggesting that
the clone B2 must have the highest sequence homology with the 3
hsp70 gene copy of the cluster (Fig. 1A). This
suggestion was further confirmed by hybridization of the 3
-UTR present
in the B2cDNA (probe B2 3
UTR-II) to the Southern blot shown in
Fig. 1C. The probe hybridized only with the 5.7-kb
SalI band, whereas it did not hybridize with the 3.8-kb
SalI repetition unit. In order to determine the copy number
of the hsp70 genes, the genomic DNA was first digested at
completion with HindIII and then partially digested with
SalI (Fig. 1D). As the Southern blot was
hybridized with the 3
UTR-II fragment of B2cDNA, the first unit of
the ladder would be 5.7 kb long and the length of the rest of the units
would be increased by 3.8 kb. Thus, since six hybridization bands
were clearly observed we concluded that at least six hsp70
gene copies should be tandemly arranged in the L. infantum
hsp70 gene cluster (Fig. 1A).
Sequence Analysis of the L. infantum hsp70 Genes
To understand in more detail the organization of the
hsp70 genes, we determined the nucleotide sequence of the
more 5 copy of the hsp70 gene cluster (gene 1) and of the
neighboring regions located at both sides of this gene. Thus, the
0.3-kb EcoRI-BamHI and the adjacent 3.8-kb
BamHI fragment, which contains the first repetition unit
present in clone B2g6 (Fig. 1A), were sequenced. A
diagrammatic representation of the sequence is shown in Fig. 2A. Table I shows the
comparison of the sequence obtained with the sequence of other
Leishmania hsp70 genes. For this analysis, the
hsp70 gene was subdivided into five regions.
|
This region extends 119 bp upstream
from the BamHI restriction site and includes the putative
spliced leader addition site. UPR regions should be present in all of
the hsp70 copies of the cluster since the UPR region of gene
1 hybridizes to each one of the genomic phages from B2g1 to B2g7
indicated in Fig. 1A. The sequence analysis indicated,
moreover, that the UPR of the hsp70 gene 1 and the UPR of
the hsp70 gene 2 show total sequence identity (Fig.
2A). As shown in Table I the L. infantum hsp70 UPR is also conserved in the hsp70 genes from other
Leishmania species. The main structural feature of the UPR
is its polypyrimidine richness (79% of C + T content) arrayed in long
pyrimidine runs. Polypyrimidine tracts have been described as a 5
essential sequence implicated in correct trans-splicing (33,
34). The spliced leader acceptor site of the L. infantum
hsp70 genes was defined by sequence similarity with those of the
L. major (18) and L. amazonensis (20)
hsp70 genes.
The 5-UTR is 161 bp long and
extends from the AG spliced leader acceptor site to the ATG
initiation codon. Comparison with the other Leishmania
species indicated that this region is also highly conserved (Table
I).
The CDR is 1959 bp long and ends just at the SalI restriction site (Fig. 2A). The derived protein product of the CDR region has been described elsewhere (24).
3The region is 1063 bp long and
extends from the TAA termination codon to the putative polyadenylation
site defined by sequence similarity with the L. amazonensis
hsp70 gene La70c1/La70gA (20). This region is highly conserved
among different Leishmania species (Table I). The nucleotide
sequence differences are mainly due to length polymorphisms of
microsatellites. In the L. infantum 3-UTR runs of
microsatellites with the sequence CA/GT are frequent. Another
remarkable feature of the L. infantum 3
UTR-I is a sequence with dyad symmetry (nucleotides 2948-3000) located next to the polyadenylation site with potential to form a stable stem-loop. Interestingly, this element is highly conserved in L. donovani and L. major hsp70 genes. Although
divergent in sequence relative to the L. infantum 3
-UTR an
equivalent stem-loop is found also in the 3
-UTR of L. amazonensis hsp70 (20).
The polyadenylation site of hsp70 gene 1 is separated from the UPR of gene 2 by 312 bp. The L. infantum IR sequence is also conserved with respect to the corresponding regions of other Leishmania species (Table I). The IR size of the Leishmania hsp70 genes is in agreement with the estimated average size (383 bp) of the IRs from other Leishmania genes (35).
As stated above, hybridization experiments (Fig. 1) indicated that the
3-UTR of hsp70 gene 6 should be different to that present
in the other hsp70 genes. The nucleotide sequence of this 3
-UTR, named 3
UTR-II, was determined on the 5.72-kb SalI
restriction fragment derived from clone B2g1 (Fig. 1A). A
diagrammatic representation of the 3
UTR-II is shown in Fig.
2B. The position of the polyadenylation site was determined
by sequence analysis of two cDNAs (called 70IIA-1 and 70IIA-2)
isolated after screening a oligo(dT)-primed cDNA library with probe
B2 3
UTR-II. The poly(A) site is placed 1059 bp downstream of the TAA
termination codon (Fig. 2B). The poly(A) site resides in
four adenosine residues. Interestingly, adenosine residues have been
described as preferential polyadenylation sites, especially when
repeated (36). Seventeen nucleotides beyond the termination codon, the
nucleotide sequences of the two 3
-UTRs start to diverge and no
sequence similarity is observed downstream. A comparative analysis
between the L. infantum 3
UTR-II and the hsp70
genes of other Leishmania species indicated that there is a
high degree of sequence similarity (85%) with the 3
-UTR of the
"orphan" hsp70 gene of L. major (18).
Remarkably, the highest values of sequence similarity between both
genes is found next to the poly(A) addition sites.
Differential Expression of hsp70 Genes
To evaluate the steady-state level of the RNA transcribed in the
hsp70 gene cluster, total RNA extracted from logarithmic phase promastigote cultures at 26 °C (normal temperature) and at
37 °C (heat shock) and from stationary phase promastigote cultures was probed with 32P-labeled oligonucleotides complementary
to specific regions of the 3UTR-I and -II (see "Experimental
Procedures" for more details). Densitometric analysis of the Northern
blots showed that the levels of expression of genes 1 through 5 increased about 2-3-fold in the parasites grown at 37 °C when
compared with those grown at 26 °C (Fig.
3A). Parasites grown in stationary phase did
not show increased levels of the genes 1-5 transcripts relative to
those found in logarithmic phase parasites (Fig. 3A). On the
other hand, the 3
UTR-II probe hybridized also with a 3.1-kb mRNA
which was found to be constitutively transcribed. The levels of these
transcripts did not change with a heat-shock treatment or in parasites
from the stationary phase (Fig. 3B). Northern blots of
poly(A)+ RNA from promastigotes in logarithmic (at 26 or
37 °C) and stationary phase probed with the different 3
-UTRs showed
a similar pattern as blots of total RNA, indicating that both
hsp70 RNAs contain poly(A) tails (not shown).
As judged by the exposure time required to have a similar
autoradiographic signal in the Northern blots hybridized with each one
of the probes, we deduced that the transcript derived from gene 6 is
more abundant than the transcripts derived from genes 1-5. In order to
quantify the abundance of the transcripts containing the 3UTR-I
(hsp70 genes 1-5) relative to those containing the 3
UTR-II
(hsp70 gene 6), a nuclease S1 protection assay was carried out using UTR-I- or UTR-II-specific oligonucleotides (Fig.
4). The densitometric evaluation of the resulting bands
showed that hsp70 3
UTR-II transcripts were about 50 times
more abundant than the hsp70 3
UTR-I transcripts from
parasites growing at 26 °C, independently of the growth phase. This
analysis indicated, moreover, that in agreement with the Northern blot
analysis shown in Fig. 3A, a heat-shock treatment promoted a
2.5-fold increase in the levels of 3
UTR-I transcripts. There were no
changes in the level of the hsp70-3
UTR-II transcripts after
a heat-shock treatment. In summary, the data show that the
hsp70 gene cluster of L. infantum contains a
hsp70 gene which is expressed constitutively and five additional hsp70 genes whose expression product is regulated
in a temperature-sensitive manner.
Post-Transcriptional Regulation of the hsp70 Gene Expression
To determine whether the abundance of the hsp70
3UTR-II transcripts relative to those containing the 3
UTR-I are due
to differential transcriptional activation, run-on transcription
analysis was carried out. The results showed that the abundance of the
nascent transcripts derived from both types of hsp70 genes
was similar (Fig. 5) as would be expected if the cluster
was transcribed as a polycistron starting at a single promotor site.
The results showed, moreover, that the temperature treatment had no
effect on the abundance of the nascent transcripts containing the
3
UTR-I region (Fig. 5) as an indication that the steady-state level of the transcripts derived from genes 1-5 must be regulated at the post-transcriptional level.
Temperature Control of hsp70 mRNA Stability
Since the differences in the steady-state level of the
hsp70-3UTR-I and hsp70-3
UTR-II transcripts
seems to be due to post-transcriptional regulation, an analysis of the
reduction of those transcripts with time after inhibition of RNA
synthesis by actinomycin D was done in Northern blots. The RNA
extracted from actinomycin D-treated cells was hybridized sequentially
with probes corresponding to the 3
UTR-I, the 3
UTR-II, the
-tubulin gene, and rDNA gene (Fig. 6). The results shown in Fig. 6 indicate that the
transcripts derived from the genes containing the 3
UTR-I are more
stable at 37 °C than at 26 °C. Densitometric analysis of the
blots indicated that 2 h of incubation at 26 °C in the presence
of actinomycin D led to reduction of the 3
UTR-I mRNA to 5% of the
transcripts present at 0 time, while after 2 h at 37 °C the
3
UTR-I transcripts were 50% of those at the 0 time. In contrast, the
transcripts derived from the gene containing the 3
UTR-II presented a
temperature-independent decay. Instead, it seems that the decay of the
3
UTR-II transcripts and those of the
-tubulin is
somewhat higher at 37 °C than at 25 °C. Also, Aly et
al. (37) reported that the L. amazonensis
-tubulin mRNA was less stable at 35 °C than at
26 °C. Hybridization of the blots to the rDNA probe indicated that
equivalent amounts of RNA were loaded in the different lanes (Fig. 6).
The comparison of the decay rates of the 3
UTR-I and -II transcripts
seems to indicate that while at 26 °C the levels of 3
UTR-I
transcripts decay faster than those containing the 3
UTR-II; at
37 °C, the decay of both transcripts is similar.
At a first view, the differences in the decay times of 3UTR-I and
3
UTR-II transcripts at 26 °C do not seem enough to explain the
differences, about 50-fold, in the steady-state RNA levels (Fig. 4).
However, we think that care should be taken in the interpretation of
these data. A densitometric analysis of the 3
UTR-II transcripts during
actinomycin D treatment indicated that they do not show a first order
decay curve; the 3
UTR-II transcripts decay slowly during the first
2 h and rapidly after that time. Thus, a secondary effect of
actinomycin D on 3
UTR-II RNA levels cannot be excluded. Hence,
alternative methods for mRNA half-time measurements are currently
used in order to determine the stability of hsp70 3
UTR-II transcripts.
Ongoing Protein Synthesis Is Required for hsp70 3UTR-I mRNA
Stabilization
In order to test for potential mechanisms of mRNA
stabilization, we examined whether the levels of decay were affected by inhibition of the protein synthesis. Thus, parallel cultures were incubated at 37 °C for different periods (0, 1, 2 and 4 h)
either in the absence or the presence of cycloheximide A in conditions in which the synthesis of protein was inhibited to 92.6% (8). At the
indicated times, culture aliquots were harvested for RNA extraction.
Total RNA samples were blotted, and the filters were sequentially
probed with the 3UTR-I and 3
UTR-II probes. Densitometric analysis of
the blots showed that in the absence of cycloheximide A the
hsp70-3
UTR-I transcripts accumulated with incubation time at 37 °C reaching a maximum after 2 h (Fig.
7A). Instead, when the same Northern blot was
hybridized with probe 3
UTR-II (Fig. 7B), it was found that
the hsp70-3
UTR-II mRNA levels remained constant along
the incubation time. However, at 37 °C and in the presence of
cycloheximide A the hsp70-3
UTR-I mRNAs did not
accumulate (Fig. 7A). The presence of cycloheximide A did
not influence the levels of the hsp70 3
UTR-II transcripts
along the heat-shock treatment (Fig. 7B). Thus, it can be
concluded that the observed stabilization at 37 °C of the 3
UTR-I
transcripts is dependent of on-going protein synthesis. Two
interpretations to these data exist: (a) a labile regulator
protein induced by heat shock should be involved in the 3
UTR-I RNA
stabilization, or (b) the 3
UTR-I RNA needs to be actively
translated in order to be stabilized by heat shock.
The heat-shock response is an evolutionary conserved mechanism that provides the cell with increased levels of a set of highly conserved proteins (hsps) which seem to be implicated in the adaptation and survival of the cell to heat and other stress conditions (38). In most of eukaryotes, hsp expression is primarily controlled at the transcriptional level. This regulation is based on a highly conserved mechanism of DNA-protein interactions between a heat-shock transcription factor and a consensus DNA sequence known as the heat-shock element (39). Given that parasitic protozoa of the Trypanosoma and Leishmania genus are subjected to a heat shock when they are transferred from the temperature of their insect vector to the 37 °C temperature of their mammalian host, it has been suggested that the heat shock may be part of a differentiation mechanism (40). Thus, heat-shock genes have been chosen as a suitable system to study gene regulation in Trypanosomatids. It has been shown that the heat-shock genes of the hsp70 and hsp83 families are transcribed constitutively at a high rate in different stages of the life cycle although the steady-state levels of the hsp transcripts increase during heat shock (16, 18, 19, 22, 23, 41-43). However, in contrast to non-protozoan eukaryotes, the cellular concentration of the hsp gene products in trypanosomes is mainly regulated at a post-transcriptional level (2-5, 37, 44, 45). It appears, moreover, that post-transcriptional regulation is common to the expression of most trypanosome genes (13).
Our data indicate that the L. infantum hsp70 gene cluster
contains, at least, six copies of the gene and that the units are arranged in tandem having conserved 5-UTRs and coding regions (Fig.
1A). However, the 3
UTR-I, common to genes 1-5, is
divergent in sequence relative to the 3
UTR-II of gene 6. In L. major four of the hsp70 gene copies are arranged in
tandem (genes 1-4), whereas the fifth hsp70 orphan gene
(gene 5) is located in a different locus (18). In the L. infantum genome the hsp70 genes 1-5 with the 3
-UTR
regions similar to those of the L. major genes 1-4, and
gene 6 with the 3
-UTR similar to that of the L. major gene 5, are located in the same cluster. Remarkably, despite the different organization of the hsp70 genes between both
Leishmania species a parallelism in the pattern of gene
expression is maintained. While the gene products of the L. major
hsp70 genes 1-4 (18) and those of the L. infantum
hsp70 genes 1-5 (this work) are increased after heat shock, the
products of the L. major hsp70 gene 5 and that of the
L. infantum hsp70 gene 6 are unaffected by temperature shifts.
Our data show also that during normal growth at 26 °C the
steady-state level of the mRNAs derived from gene 6 is 50-fold
higher than the level of the mRNAs derived from genes 1-5 and that
only the expression of the genes 1-5 increased after a heat-shock
treatment while the expression level of gene 6 remained unaffected. The results of the run-on assays showed that all genes are transcribed at
similar rates before and after heat shock as it would be expected from
the present of conserved 5-UTRs and intergenic regions along the
cluster. The results are also in agreement with a polycistronic transcription of all the genes of the cluster from an unknown promotor.
Our data also showed that at 37 °C the hsp70-3UTR-I
transcripts are more stable at 37 °C than at 26 °C but that the
heat-shock treatment did not affect the hsp70-3
UTR-II
mRNA stability. Thus, the mechanisms responsible for the
preferential accumulation of L. infantum hsp70-3
UTR-I
mRNAs upon temperature elevation must be related with the sequence
divergence between the two types of 3
-UTRs. Remarkably, the 3
UTR-I of
genes 1-5 contains, next to the putative polyadenylation site, an
inverted repeat with potential to form a stable stem-loop structure
that might be implicated in the stabilization of the
hsp70-3
UTR-I transcripts. A potentially similar stable
stem-loop structure is absent in the 3
UTR-II of gene 6. In other
eukaryotes, in addition to transcriptional activation, post-transcriptional regulation of hsp70 genes has been also
observed. For example, it has been reported that the Drosophila
hsp70 mRNA is rapidly degraded at normal temperatures and
stabilized by heat shock and that the regulatory mechanism operates
through recognition of the 3
-UTR of the hsp70 mRNA
(46). The question, however, of whether the Drosophila hsp70
mRNA is an inherently stable message that is selectively degraded
at normal temperatures or it is an inherently unstable message that is
stabilized by heat shock remains open (47). In humans cells, it was
observed that the heat shock increases the HSP70 mRNA
stability at least 10-fold and that the HSP70 mRNA is
more stable in cells treated with protein synthesis inhibitors
suggesting that a heat shock-sensitive labile protein regulates its
turnover (48). The effect of the heat shock on the L. infantum
hsp70-3
UTR-I mRNA levels is similar to the effect of the
treatment on the L. amazonensis hsp83 mRNA levels (3). However, the mechanism responsible for the temperature-induced accumulation of the L. infantum hsp70 mRNAs seems to be
different from the one responsible for the regulation of L. amazonensis hsp83 mRNAs. The degradation of hsp83
mRNAs in L. amazonensis depends on active protein
synthesis suggesting the implication of a labile nuclease that is
active mainly at 26 °C (3). Our results indicated, however, that in
the presence of cycloheximide at 37 °C the levels of the
hsp70 3
UTR-I transcripts remain constant while in the
absence of the drug the levels of the transcripts increased as an
indication that ongoing protein synthesis is required to attain their
stability. A model which might explain this result would implicate the
direct interaction of a labile protein factor with the 3
UTR-I of the
mRNA hindering the activity of the nucleases implicated in mRNA
degradation pathways. An alternative model would invoke the presence of
a labile protein factor which can promote a down-regulation of a
specific nuclease for the hsp70-3
UTR-I transcripts. In both
models the putative labile protein factor would be active mainly at
37 °C. At present, a search for this putative protein factor is
underway.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y08019[GenBank] and Y08020[GenBank].
We thank Dr. E. Rondinelli for the T. cruzi -tubulin cDNA (clone pTc
3). We are also grateful
to Dr. J. P. García-Ruiz for assistance in run-on assays.