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INTRODUCTION |
Leishmania is a protozoan parasite that infects and
replicates within mammalian macrophages, thereby causing a wide
spectrum of diseases in humans. The parasite's life cycle comprises
two stages involving two different hosts: the flagellated promastigote stage, which is present in the gut of the sand fly vector, and the
aflagellated non-motile amastigote present in the phagolysosomes of
mammalian macrophages. During its developmental cycle,
Leishmania adapts to the different environments encountered
by undergoing a series of morphological and biochemical changes
mediated by the differential expression of a variety of genes. Until
now, only few of these genes have been identified, and the mechanisms of their stage-specific regulation are still poorly understood.
In Leishmania, several studies have shown that
post-transcriptional regulation of developmentally expressed
transcripts involved sequences present mainly in the 3'-untranslated
regions (1-3) but also in 5'-untranslated regions (1) and in
intergenic regions between tandemly repeated genes (4, 5). Similarly,
in trypanosomes, elements in the 3'-untranslated and intergenic regions
of many mRNAs were found to be involved in the post-transcriptional
regulation of gene expression, RNA stability, or translation (6-10).
The predominance of post-transcriptional over transcriptional controls is probably because of the organization of the trypanosomatid genome in
large polycistronic transcription units (11-14). Maturation of all
pre-mRNAs in these organisms involves two RNA processing reactions; trans-splicing (15, 16) and 3'-end cleavage linked to
polyadenylation (17, 18). Polycistronic precursor RNAs are
processed into monocistronic units by the excision of intergenic sequences and the addition of a 5'-capped 39-nucleotide spliced leader
(SL1 or mini-exon) to the
5'-end of each pre-mRNA by trans-splicing. The mini-exon sequence
is donated from a small independent RNA, the SL RNA or
mini-exon-derived RNA, which is required for RNA processing, transcript
stability, and initiation of translation in a variety of trypanosomatid
protozoa (16). Trans-splicing constitutes an essential step in
transcript's maturation and, as such, may serve as an important mode
for the control of gene expression in kinetoplastida. It has been
previously shown that exposure of Trypanosoma brucei
procyclics to severe heat shock inhibited trans-splicing and processing
of the
- and
-tubulin transcripts (19).
In this study we report that the SL RNA of Leishmania
donovani is developmentally regulated in the amastigote stage.
This regulation involves a specific polyadenylation of a longer SL transcript, which seems to be synthesized specifically from one class
of the genomic mini-exon repeats. This is the first time where
polyadenylation of a normally non-polyadenylated RNA is reported as
part of a putative regulatory mechanism aiming at controlling gene
expression in a specific stage of Leishmania's life cycle.
Specific polyadenylation of the SL RNA may represent an additional step
in the gene regulation process during Leishmania differentiation into the amastigote form.
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EXPERIMENTAL PROCEDURES |
Parasite Strains and Culture Media--
L. donovani
donovani Sudanese 1S2D and L. donovani infantum LEM1317
(from Dr. Bastien, Montpellier, France) strains were maintained in RPMI
1640 medium supplemented with 10% heat-inactivated calf serum and 5 mg/ml hemin. Promastigotes were cultured in RPMI at pH 7.0 and
25 °C. To induce axenic differentiation we used a slightly modified
protocol published by Joshi et al. (20) where stationary phase parasites were transferred into RPMI medium supplemented with
20% calf serum and MES to maintain the pH at 5.0 and grown for 5 days
at 25 °C before being shifted to 37 °C at a 5% CO2 atmosphere for an additional 24 h. Amastigotes were also isolated from the spleens of gold Syrian hamsters infected intraperitoneally with 5 × 107 L. donovani 1S2D
amastigotes as described (21).
Nucleic Acid Preparations and Analyses--
Total RNA of
L. donovani promastigotes and amastigotes was isolated using
the guanidinium isothiocyanate method with TRIzol (Life Technologies,
Inc.). cDNAs corresponding to all poly(A)+ mRNA
transcripts were synthesized using an oligo(dT)18 primer and the Superscript II reverse transcriptase (Life Technologies, Inc.).
Southern and Northern blot hybridizations were performed following
standard procedures (22). The two probes used for these studies
correspond to the intron sequence of the spliced leader RNA gene
generated by PCR using P1 (5'-ATTGGTATGCGAAACTTCCG-3') and P2
(5'-CCGCTACGGAAGCCGCCGGC-3') primers (see Fig. 8) and to the whole
mini-exon repeat, as part of the 434-bp NcoI fragment. Neomycin phosphotransferase (neo) and hygromycin
phosphotransferase (hyg) specific probes were made by PCR
and used for determining the vector copy number in the transfectants.
The L. donovani infantum LEM1317 cosmid library was prepared
by ligating size-selected Sau3AI partial digest products of
genomic DNA with the shuttle vector cL-HYG (23), cleaved by
BamHI, and packaged using the Gigapack Gold II kit
(Stratagene). The library was gridded onto nylon membranes at the
Toronto Genome Center (Hospital for Sick Children) to generate high
density filters of 92 × 4 × 4. High density filters of the
same series were hybridized independently to L. donovani
amastigote and promastigote 32P-labeled cDNA probes.
Recombinant DNA Constructs and Transfections--
Constructs
pSP
neo
-miniexA, pSL
neo
-miniexB, and pSL
neo
-miniexC
bearing the three different mini-exon genomic copies of L. infantum LEM1317 were made as follows. The 424-bp NcoI
fragments (copies B and C) from cosmid 3H4 (carrying ~20 kilobase
pairs of the mini-exon locus) were subcloned into the NcoI
site of pSL1180 (Amersham Pharmacia Biotech), and the
SmaI-BamHI
neo
expression cassette (24)
from vector pSP72
neo
was introduced into
EcoRV-BamHI sites of pSL1180 to generate
pSL
neo
-miniexB and pSL
neo
-miniexC, respectively. The 434-bp
NcoI fragment (copy A) was subcloned into the
BamHI site of pSP72
neo
expression vector following a
Klenow polymerase treatment. Expression of the neo gene in
pSP72
neo
and pSL
neo
is driven by the intergenic region of
the
-tubulin gene (represented here by
) (25). The cDNAs of
the poly(A)+ and poly(A)
SL transcripts (see
below) were amplified by RT-PCR using P1 (see Fig. 8) and an
oligo(dT)18 as primers and subcloned into pCRTM
2.1 (Invitrogen) to generate pSL-RNA(A)+ and
pSL-RNA(A)
vectors, respectively. The nucleotide sequence
of the different genomic mini-exon repeats of L. donovani
1S2D and L. infantum LEM1317 and of the cDNAs was
determined by an ABI Prism 377 DNA automated sequencer. 10-20 µg of
plasmid or cosmid DNAs were used for transfections into L. donovani by electroporation as described previously (24).
Nuclear Run-on and RNA Stability Assays--
Nuclear run-on
assays were performed essentially as described by Quijada et
al. (3) using nuclei freshly isolated from L. donovani
exponential phase promastigote and amastigote cultures. To look for
RNA stability, late-log phase promastigotes were submitted to growth
conditions mimicking those found in the phagolysosomes at pH 5.0 for 5 days followed by 1-2 h of incubation at 37 °C in the presence of
5% CO2, prior to the addition of 10 µg/ml actinomycin D
(Sigma). Cells were harvested by centrifugation, and total RNA was
extracted at different time points of actinomycin D treatment: 0, 30 min, 1, 2, 4, and 8 h. Total RNA samples were subjected to
Northern blot analysis using the intron sequence of the mini-exon gene
as a probe. Quantitation of the SL transcripts was done by densitometric analysis using a PhosphorImager with the ImageQuant 3.1 software.
Mapping of the 3'-End of Poly(A)
and
Poly(A)+ SL RNAs--
To map the 3'-end of the
polyadenylated SL RNA, a double-stranded cDNA was synthesized from
axenic amastigotes using P1 and an oligo(dT)18 as primers.
3'-RACE studies were used to map the 3'-end of the non-polyadenylated
SL transcript. A poly(A) tail was added at the 3'-end of this
transcript following incubation of 10 µg of total L. donovani promastigote RNA with 5 units of poly(A) polymerase (Life
Technologies, Inc.) according to the manufacturer's recommendations.
RT-PCR reactions were carried out using the above set of primers. The
PCR products were then gel-excised, further purified using
phenol/chloroform extraction, subcloned into the pCRTM 2.1 vector, and sequenced.
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RESULTS |
Differential Expression of the SL RNA Gene in L. donovani
Amastigotes--
To identify genes that are developmentally regulated,
we developed a genomic differential screening approach where high
density filters, each covering approximately one parasite genome, were used. A genomic cosmid library made from L. donovani
infantum strain LEM1317 was gridded onto high density filters
(1536 cosmid clones/filter), which were hybridized with
32P-labeled cDNA probes derived from promastigote and
amastigote RNAs. Comparative analysis of the hybridization signal
intensity between the promastigote and amastigote cDNA probes in
four series of high density filters allowed the identification of a
number of cosmid clones that were much more expressed in amastigotes. Normalization of the amount of bacteria layered at each position was
obtained by hybridization with a vector-specific probe
(hyg). Among a large number of clones selected on the basis
of their higher expression in amastigotes, 10 were related. Partial
sequence analysis of these clones indicated that we were dealing with
the spliced leader RNA gene locus. From those 10, cosmid 3H4 was chosen for further analysis (Fig.
1A). Southern blot
differential hybridization of cosmid 3H4 using promastigote and
amastigote cDNA probes revealed the presence of a 434-bp
NcoI genomic fragment hybridizing much stronger to the
amastigote cDNA (Fig. 1B). The nucleotide sequence of
this fragment confirmed the presence of the mini-exon gene repeat.

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Fig. 1.
Differential genomic screening for the
isolation of amastigote-specific clones. A,
differential hybridization of one set of high density filters of the
L. donovani infantum LEM1317 cosmid library with
promastigote (Pro) and amastigote (Am) cDNA
probes. The arrow indicates cosmid 3H4 selected for these
studies on the basis of its higher expression in amastigotes.
B, Southern blot hybridization of cosmid 3H4 with
promastigote and amastigote cDNA probes. The arrow
pinpoints a ~434-bp NcoI fragment showing a differential
hybridization signal. This fragment contained the mini-exon repeat as
demonstrated by sequence analysis.
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Total RNA preparations from promastigotes and axenic amastigotes were
hybridized with a probe corresponding to the intron sequence of the
L. infantum mini-exon repeat that we have sequenced. Two
transcripts of different sizes were detected in the amastigote RNA
preparation, one corresponding to the expected ~86-nt SL RNA and a
second larger transcript of ~170 nt (Fig.
2A). In addition, a 2.5-fold
increase in the 86-nt SL transcript was observed in the amastigote RNA
preparation (Fig. 2A). To determine whether this
developmental regulation of the SL transcript was in fact a
physiological event and was not because of an artifact due to the
axenic conditions used for parasite differentiation, we tested amastigotes freshly isolated from infected hamsters. As shown in Fig.
2B, a larger size SL transcript was detected in the RNA preparation from hamster-derived amastigotes similar to what was seen
when using the axenic amastigotes. The larger SL transcript was
estimated, by densitometric analysis using PhosphorImager scanning of
several independent Northern blots, to represent 12-16% of the total
SL RNA synthesized within the amastigote cell.

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Fig. 2.
Expression of the SL RNA in L. donovani promastigotes and amastigotes. A,
Northern blot analysis of total RNA isolated from L. donovani 1S2D promastigotes (Pro) and axenic
amastigotes (Am). Equal amounts of total RNA were
transferred into a nylon membrane and hybridized to a spliced leader
intron probe. Hybridization with the A2 amastigote-specific
gene (21) was served as an internal control for the axenic
differentiation. B, expression of SL RNA in L. donovani amastigotes isolated from infected spleens of gold Syrian
hamsters. The bottom panel represents an ethidium
bromide staining of the RNA samples loaded on agarose gel.
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Stage-specific Expression of the SL RNA Is Induced upon Growth at
Acidic Conditions--
During their life cycle Leishmania
are exposed to temperature and pH changes that trigger stage-specific
differentiation (26). Elevated temperature or acidic pH has been
associated with the induced expression of a number of genes (21, 27,
28). To assess the role of pH or of temperature in the differential
regulation of the SL RNA, different culture conditions were tested.
L. donovani promastigotes were subjected to a pH shift
from pH 7.0 to 5.0 for 5 days at 25 °C or to a temperature shift at
37 °C in a 5% CO2 environment, or to both. Total RNA
was extracted at different time points and analyzed by Northern blot
using the PCR-amplified spliced leader intron sequence as a probe. As
shown in Fig. 3 acidic pH seems to be the
main factor that induces the expression of the larger ~170-nt SL
transcript but is also responsible for the increased levels of the
~86-nt SL RNA. This pH-dependent induction of the SL
transcripts occurs rapidly within the first hours of incubation under
acidic conditions (Fig. 3 and not shown). Developmental regulation of
the SL RNA is specific to the amastigote stage because no larger
transcript or any significant increase in the SL RNA levels were
observed in stationary or metacyclic L. donovani cultures (Fig. 3).

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Fig. 3.
Differential expression of the SL RNA upon
growth of the parasites at acidic pH conditions. Total RNA (10 µg) was extracted from L. donovani 1S2D cultured under
different conditions to obtain promastigotes (Pro),
metacyclics (Met), and axenic amastigotes where shifts in pH
or in temperature, or both, were independently tested. All the above
RNAs were analyzed by Northern blot using the intron sequence of the
mini-exon gene as a probe (top panel). The
bottom panel shows ethidium bromide staining of
the RNA samples loaded on agarose gel.
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Differential Regulation of the SL RNA Is Because of a Specific
Polyadenylation in Amastigotes--
We hypothesized that the larger
170-nt SL RNA present in amastigotes might be the result of an
alternative 3'-end formation or of a post-transcriptional modification
of the transcript. Several previous studies have reported that the
kinetoplastid SL RNA, as for most of the SL RNAs in nematodes (29), is
not polyadenylated and that it is constitutively expressed throughout
the developmental stages of these parasites (30, 31). However, the SL
transcript has been detected within the poly(A)+ RNA
fraction of T. brucei bloodstream forms (32). To test
whether polyadenylated SL RNA could also be found in
Leishmania, we first synthesized cDNAs from total RNA of
L. donovani promastigotes and axenic amastigotes using and
oligo(dT) primer, allowing only the polyadenylated RNAs to be reverse
transcribed, and hybridized them with the mini-exon repeat as a probe.
Because of the presence of the 39-nt spliced leader at the 5'-ends of
all pre-mRNAs, a hybridization smear corresponding to all
polyadenylated transcripts was observed in both promastigote and
amastigote cDNAs (Fig.
4A). A cDNA fragment
corresponding to the SL RNA gene was found exclusively in the
amastigote cDNA preparation, strongly suggesting that
polyadenylated forms of the SL RNA could indeed be present in
Leishmania amastigotes (Fig. 4A). To verify
whether this poly(A)+ SL transcript was also found in
L. donovani amastigotes isolated from infected hamsters,
similar experiments were performed, and they gave rise to identical
results (data not shown). The presence of the poly(A)+ SL
transcript in amastigotes was confirmed by RT-PCR studies where a
cDNA synthesized from a poly(A)+ fraction was subjected
to PCR amplification using a combination of P1 (see Fig. 8) and
oligo(dT) primers to amplify part of the SL transcript that lacks the
common 39-nt spliced leader. A ~130-bp cDNA fragment amplified by
PCR and corresponding to the poly(A)+ SL RNA was detected
by hybridization only in the amastigote poly(A)+ fraction
(Fig. 4B). PCR products hybridizing to the intron probe were
extracted from the agarose gel and subcloned into vector pCRTM 2.1. Sequence analysis definitively confirmed
the poly(A)+ addition in the SL transcript expressed
specifically in the amastigote stage (Fig.
5A).

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Fig. 4.
Detection of the SL transcript in the
poly(A)+ RNA fraction of L. donovani
amastigotes. A, hybridization of cDNAs
synthesized from promastigote (Pro) and amastigote
(Am) total RNA with the mini-exon repeat as a probe. The
arrow indicates the differentially expressed SL transcript
present in the poly(A)+ fraction of the amastigote
cDNA. B, promastigote and amastigote cDNAs of
L. donovani synthesized from poly(A)+ fractions
were amplified by PCR using P1 (beginning of the intron sequence) and
oligo(dT) as primers and hybridized with the spliced leader intron
probe. Vector pSL-RNA(A)+ carrying the cDNA sequence
corresponding to the ~170-nt SL RNA and the genomic L. donovani DNA and vector pSP72 DNA (control) were served as
controls for the PCR reactions.
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Fig. 5.
Sequence analysis of the poly(A)+
and poly(A) SL transcripts. A. Alignment
of the nucleotide sequences of the poly(A)+ and
poly(A) SL RNAs. The 39-nt spliced leader sequence common
to all trypanosomatid pre-mRNAs is underlined. The
poly(A)+ SL RNA contrary to the poly(A)
transcript contains an additional 15-nt sequence at its 3' end followed
by a long poly(A) tail (>46 As). B-C. Predicted
secondary structures of the poly(A) (B) and
poly(A)+ (C) SL RNA folded using the computer
algorithm (FOLD). The graphic representation of the secondary structure
was produced using the SQUIGGLES program. An estimate of the free
energy required for structure formation is calculated at 18.8
kcal/mol for the poly(A) SL transcript and at 21.2
kcal/mol for the poly(A)+ SL RNA.
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To compare the 3'-end of the poly(A)+ and
poly(A)
SL transcripts, we also mapped the 3'-end of the
normally non-polyadenylated transcript in our L. donovani
1S2D strain by a 3'-RACE assay. The L. donovani
poly(A)
SL RNA ended at position 86 (Fig. 5A),
slightly different from what has already been published for other
Leishmania species where the 3'-end was mapped at nucleotide
89 (31) or nucleotide 96 (33). Sequence comparison between the
cDNAs corresponding to the polyadenylated and non-polyadenylated SL
transcripts revealed a significant difference at their 3'-ends. Indeed,
the poly(A)+ SL RNA contained 15 additional nucleotides,
ending by four Ts part of the 8T-track sequence (see Fig. 8) in
comparison with the poly(A)
transcript (Fig.
5A). A long poly(A) tail of at least 46 As was added
immediately after the four Ts. Considering that the
poly(A)+ SL RNA is ~170 nt long, the poly(A) tail could
include as much as 80 As. Comparison of the predicted secondary
structures of the poly(A)
and poly(A)+ SL
RNAs using the SQUIGGLES program did not suggest a significant overall
change in the free energy required for structure formation (
G), but it revealed the presence of a third stem loop
structure at the 3'-end of the SL poly(A)+ RNA (Fig.
5B), which could possibly be a substrate for RNA binding proteins.
Polyadenylation of SL RNA Increases Its Stability--
The effect
of pH on the stability and accumulation of the SL transcripts was
addressed. We first evaluated, by run-on experiments, whether the
increase in poly(A)
SL RNA accumulation or the presence
of the poly(A)+ SL transcript in amastigotes (see Figs. 2
and 3) was because of higher SL RNA synthesis rates in this parasite
stage. Nuclei were freshly isolated from promastigotes, and axenic
amastigotes and radiolabeled nascent RNAs were extracted and used to
probe the mini-exon and control DNA sequences. No differences were
observed in the overall synthesis of SL RNA between promastigotes and
amastigotes (Fig. 6), suggesting that the
regulation occurs post-transcriptionally. Although secondary structure
predictions did not suggest that the addition of the 15 nucleotides at
the 3'-end of the larger SL RNA should increase its stability
significantly (see Fig. 5B), it is, however, known that
polyadenylation in general increases message stability (for review see
Ref. 34). We therefore looked to see whether the poly(A)+
SL RNA was more stable compared with the poly(A)
transcript. Because we showed that the maximum accumulation of the
polyadenylated SL RNA is observed in cells maintained at pH 5.0 (Fig.
3), we evaluated the stability of the SL transcripts under these growth
conditions. In these assays, L. donovani promastigotes were
transferred from pH 5.0 and 25 °C to 37 °C for 1 h to adapt in these conditions prior to the addition of 10 µg/ml actinomycin D,
an inhibitor of RNA synthesis. Total RNA samples were extracted at
different time points following the addition of actinomycin D to the
culture medium and analyzed by Northern blot using the spliced leader
intron as a probe. The relative amount of the poly(A)+ and
poly(A)
SL transcripts present in the cell at each time
point was compared with the RNA levels present at time 0. Quantitative
evaluation of the SL RNA levels obtained by densitometric analysis of
radiolabeled membranes using a PhosphorImager demonstrated that the
poly(A)+ SL RNA was 2.5-3-fold more stable compared with
the non-polyadenylated transcript (Fig.
7, A and B).
Similar results were obtained from two more independent experiments.
The small shortening of the poly(A)+ SL RNA observed over
time may correspond to differences in RNA migration in that part of the
gel (not shown). In light of these results, the half-lives were
established at 4 h for the poly(A)+ SL RNA and at
2 h for the poly(A)
transcript.

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Fig. 6.
Analysis of nascent SL RNA transcription in
L. donovani promastigotes and amastigotes.
[32P]UTP nascent RNAs isolated from nuclei of L. donovani promastigotes (Pro) and amastigotes
(Am) were hybridized to nylon membrane-bound single-stranded
DNA probes from the mini-exon repeat and the ribosomal 18 S DNAs.
Vector pGEM-7Zf was used as a negative control.
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Fig. 7.
Relative stability of the
poly(A)+ SL RNA versus the
poly(A) transcript. A, total RNA was
isolated from L. donovani 1S2D axenic amastigotes after
different incubation times with actinomycin D to inhibit RNA synthesis.
RNA aliquots (15 µg) were applied to a 2% agarose gel, transferred
to a nylon membrane, and hybridized with the spliced leader intron
probe. Dimethyl sulfoxide (DMSO, the solvent for actinomycin
D) was added for 8 h to one sample as a control. RNA loading was
monitored by ethidium bromide staining of the gel prior to transfer
(bottom panel). Pro, promastigotes.
B, quantitative evaluation of the SL RNA levels shown in
A was obtained by scanning densitometry of the radiolabeled
membrane using a PhosphorImager. Values are presented as a histogram
and expressed as a percentage of the hybridization signal intensity of
both the polyadenylated and the non-polyadenylated SL transcripts for
each time point compared with time 0. Stability assays were repeated
three times giving rise to similar results.
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Stage-specific Polyadenylation of One Class of the Mini-exon
Genomic Copies--
To examine whether differences within the
non-transcribed spacer of the mini-exon genomic copies could account
for the formation of the new 3'-end of the poly(A)+ SL RNA,
the nucleotide sequence of several copies was determined. Several
~434-bp NcoI copies derived from cosmid 3H4 were subcloned into pSP
neo
and PSL
neo
expression vectors, and their
nucleotide sequence was compared. By compiling the sequencing data from
a large number of clones (~20), we classified the mini-exon gene repeats into three classes (A-C). The alignment of the nucleotide sequences of these three different copies along with a previously published copy derived from another L. infantum strain is
presented in Fig. 8. As expected, the
39-nt spliced leader was highly conserved, whereas three nucleotide
changes and one short deletion were observed in the intron sequence of
the two L. infantum strains. However, considerable
differences were noted within the non-transcribed spacers of the two
L. infantum strains (see Fig. 8). Analysis of the three
copies of the LEM1317 strain showed only one difference located between
nucleotides 140 and 150. Copy A indeed contains a 10-bp GC-rich
stretch, which is absent in copies B and C, where only a single base
pair mismatch from a T to C at position 150 distinguishes these two
copies (Fig. 8).

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Fig. 8.
Sequence alignment of the three L. donovani infantum LEM1317 mini-exon gene copies. The
~434-bp NcoI fragments derived from cosmid 3H4 were
sequenced, and copies A, B, and C, which can be found under the
accession numbers AF097653, AF097654, and AF097655, respectively, were
aligned together with the mini-exon repeat of another L. donovani
infantum MHOM 80 strain. The 39-nt common spliced leader is
underlined. Open boxes indicate the
differences depicted between the nucleotide sequences of the mini-exon
gene repeats. Primers P1 and P2 used for PCR and
RT-PCR studies are also indicated.
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We tested whether one of these genomic copies was associated with the
larger ~170-nt SL transcript. Accordingly, we made the appropriate
constructs to overexpress each one of the three mini-exon gene repeats
(A-C) in L. donovani (Fig.
9A). We also transfected cosmid 3H4, which contains the three different classes of the mini-exon
repeats. Stable transfectants were subjected to differentiation from
promastigotes to axenic amastigotes. RNA samples from wild type and
transfectants were analyzed by Northern blot using a probe
corresponding to the spliced leader intron sequence. A significant increase in the poly(A)+ SL transcript was detected only in
the transfectants expressing copy A or cosmid 3H4, respectively (Fig.
9B). No differences between the transfectants carrying copy
B or C and the wild-type strain were observed, suggesting a certain
specificity of the polyadenylation process. To ascertain these results
we compared the copy number of the expression vectors in the
transfectants by hybridization studies using neo and
hyg genes as probes. No significant differences in the copy
number of the three mini-exon repeats were noted between transfectants
(Fig. 9C), thus supporting further the implication of copy A
in the formation of the longer SL transcript that becomes polyadenylated. By random sequencing of a large number of clones derived from cosmid 3H4 we roughly estimated that each class is equally
represented in this cosmid.

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Fig. 9.
Stage-specific polyadenylation of one class
of the mini-exon genomic copies. A, schematic
representation of the constructs used for the episomal expression of
the L. infantum LEM1317 mini-exon gene repeats
(A-C) into L. donovani. The expression of
neo in these vectors is driven from the intergenic region of
the -tubulin gene (IR- ). B, Northern blot
hybridization of mini-exon recombinant transfectants (3H4,
A, B, C) and wild-type cells subjected
to an axenic differentiation with the spliced leader intron probe.
Pro, promastigotes; Am, amastigotes.
C, estimation of the mini-exon repeats copy number in the
transfectants shown in A by hybridization studies.
Densitometric analysis was carried out as described in Fig.
7B, and the quantitation of the hyg- or
neo-containing vectors was based on comparison studies with
single integration of the neo and hyg genes
within the parasite genome.
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DISCUSSION |
Using a genomic differential screening approach based on the
hybridization of high density filters with promastigote and amastigote cDNA probes, we have isolated a number of differentially expressed cosmid clones that contained several mini-exon gene repeats. In kinetoplastida, the SL RNA is encoded by a cluster of ~150-200 tandemly repeated copies per genome. Each repeat comprises a highly conserved 39-bp exon sequence, a moderately variable (55-101 bp) intron sequence, and a non-transcribed highly variable spacer between
different genus and species (35-38). The spliced leader, a common
39-nucleotide sequence, is transferred via trans-splicing from the
precursor SL RNA to the 5'-end of all trypanosomatid pre-mRNAs.
We have shown that stage-specific regulation of the SL RNA in both
L. donovani and L. infantum is mediated by a
specific polyadenylation process. Upon growth of the parasite at acidic
pH, 12-16% of the total SL RNA becomes polyadenylated, giving rise to
a larger 170-nt transcript in addition to the 86-nt mature SL RNA
(Figs. 2, 3, and 5A). A temperature shift alone
(25-37 °C) was not capable of inducing the expression of the
polyadenylated SL RNA or of increasing the amount of the
non-polyadenylated transcript (Fig. 3). Several reports have confirmed
the constitutive expression of the SL RNA in all developmental stages
of trypanosomes and Leishmania, and it is generally assumed
that the SL RNA is not polyadenylated (30, 31). Similarly, spliced
leader RNAs in the nematode Caenorhabditis elegans are
normally not polyadenylated although one case has been reported (29).
Polyadenylated forms of the SL transcript have been identified in
poly(A)+ RNA fractions from short stumpy bloodstream forms
of T. brucei, however (32).
Mapping of the 3'-end of the poly(A)
and
poly(A)+ SL RNAs revealed that the poly(A)+ SL
RNA was longer by 15 nucleotides compared with the 86-nt
non-polyadenylated SL transcript (Fig. 5A). Little is known
about the process of trypanosomatid mRNA 3'-end formation. Recent
data reported that transcription termination of the
Leishmania tarentolae SL RNA is mediated by the
downstream T-track at the base of stem-loop III (39). We have shown
that an alternative maturation site was used by the
poly(A)+ SL RNA and that this transcript was ended by four
Ts as part of an 8Ts track sequence in the mini-exon gene repeats
present in our strain (Figs. 5A and 8). We have also
demonstrated that only one of the three different classes of the
mini-exon gene in L. infantum, the mini-exon copy A, showed
a significant increase in the levels of the poly(A)+ SL
transcript under pH stress at relatively similar rates as those of
cosmid 3H4 carrying the three classes of the mini-exon genomic copies
(Fig. 9B). These results support the implication of this
particular mini-exon copy as a template for the formation of the longer
170-nt SL transcript. It is not clear, however, how the 10-bp GC-rich
motif present only in the non-transcribed spacer of copy A between
nucleotides 140 and 150 could influence maturation of the SL RNA. It is
possible that changes in the secondary structure could allow the
polymerase to pause further downstream. Alternatively, it is possible
that the mini-exon copy A is transcribed by a different polymerase.
Reports on the type of polymerase that transcribes the mini-exon gene
of kinetoplastid protozoa have yielded conflicting data. It has been
reported that the mini-exon repeats are transcribed by polymerase II
(40, 41), but other studies support that they are transcribed by
polymerase III (42). It is assumed from several studies that the
tandemly repeated mini-exon genes in trypanosomatids are independently
transcribed (41, 43), and this is in line with the possibility that
some mini-exon copies could be transcribed by different polymerases.
It is assumed that the 3'-end of mature mRNAs is generated by a
cleavage of the pre-mRNA at the polyadenylation site followed by
the addition of the poly(A) tail (44). No conserved sequence motif like
the AAUAAA of higher eukaryotes has been identified, however, upstream
of the kinetoplastid mRNA polyadenylation sites, suggesting a
mechanism different from the one used in other systems (15). One
important factor determining the site of polyadenylation in
Leishmania is the positioning of a functional splice
acceptor site immediately downstream as polyadenylation and
trans-splicing seem to be coupled in this organism (17). Accurate
polyadenylation in trypanosomatids is often determined by
pyrimidine-rich elements in the intergenic regions (45-47).
Polypyrimidine tracts are recognized by both the trans-splicing and
polyadenylation machineries either sequentially or simultaneously (18).
The additional 15-nt sequence part of the very 3'-end of the
poly(A)+ SL RNA is rich in pyrimidines (10 nucleotides of
15), and it contains an AG splice acceptor site immediately preceded by
a pyrimidine tract (5'-AGGCGCTCTTTT-3') (see Fig. 5A). This
splice acceptor site may be recognized by splicing factors that could activate polyadenylation. This has been seen in higher eukaryotes (48).
Although polyadenylation has been reported in bacteria and some plastid
mRNAs to accelerate transcript degradation (49, 50) it is generally
assumed that in most eukaryotic systems the poly(A) tract protects
mRNAs from rapid degradation (51-53). In addition, the poly(A)
tail influences nuclear processing of the pre-mRNA,
nucleocytoplasmic transport, and translation (34, 53-55).
Polyadenylation of the SL RNA in Leishmania increases its stability by a factor of 2.5-3 as estimated from the actinomycin D
data (Fig. 7). The presence of the 15-nt additional sequence at the
3'-end of the poly(A)+ SL RNA does not seem to introduce
any significant change in the
G (from
21.2 to
18.8
kcal/mol) required for RNA structure formation, but it allows the
formation of a stem-loop structure at the 3'-end of the transcript
(Fig. 5B). Stem-loop structures at the 3'-ends of many
transcripts function as binding sites for regulatory proteins that
could affect RNA transport, translation, and half-life (34). It is
possible that the poly(A)+ SL RNA is exported into the
cytoplasm as part of a regulatory mechanism to decrease the overall
levels of SL RNA in the nucleus of the amastigote cells. Amastigotes
replicate more slowly than promastigotes, and it is likely that less SL
RNA is required for mRNA processing by trans-splicing. This is in
line with our observation that even the poly(A)
SL RNA
accumulates in the amastigote stage. Indeed, a 2.5-fold increase in SL
transcript levels was seen during differentiation of the parasite into
the amastigote form (Figs. 2 and 3). The half-life of transcripts could
change in response to nutrient levels, cell growth rates, and
temperature shifts, among other things (56), and this could also be the
case for the SL RNA under acidic stress. It is also possible that the
polyadenylated SL transcript cannot be used as a donor of the 39-nt
spliced leader sequence to the 5'-end of all pre-mRNAS. As a
consequence, this should decrease trans-splicing reactions in
amastigotes by ~15%. It has been shown that L. tarentolae
SL transcripts that were malformed by more than 1 nucleotide at their
3'-ends were unable to participate in trans-splicing or acquiring a
proper cap 4 methylation (39).
During their life cycle Leishmania protozoans alternate from
the promastigote to the amastigote stage, undergoing several morphological and biochemical changes that are monitored by the differential expression of a variety of genes. Previous studies have
reported amastigote-specific regulation of a number of genes: hsp83 (27), hsp100 (57), histone H1 (58),
cpb cysteine proteinases (59), A2 gene family (21), and the
LmcDNA16 gene family (60). Our studies demonstrated that part of
the spliced leader RNA in L. donovani amastigotes is
polyadenylated. This polyadenylation is stage-regulated and may
correspond to a general post-transcriptional mechanism operating upon
parasite differentiation to control gene expression.