(Received for publication, April 21, 1995; and in revised form, July 12, 1995)
From the
The genome of Trypanosoma cruzi contains tandemly arrayed copies of the gene encoding amastin, an abundant protein on the surface of the amastigote stage of the parasite. The transcription rate of the amastin genes is the same in the different developmental stages, but the steady state level of the 1.4-kilobase amastin mRNA is 50-85 times higher in amastigotes than in epimastigotes or trypomastigotes(1) . Here we show that the amastin genes alternate with genes encoding another protein, called tuzin, whose 1.7-kilobase mRNA is much less abundant in amastigotes. The 3`-untranslated region (UTR) of tuzin mRNA is only a few nucleotides in length or even nonexistent, in contrast with the 630-nucleotide 3`-UTR of amastin mRNA. No promoter elements were found upstream or within the amastin/tuzin gene cluster. However, in amastigotes, the protein synthesis inhibitor cycloheximide caused a 3-fold decrease in amastin mRNA and a 7-fold increase in tuzin mRNA. Furthermore, when the amastin 3`-UTR plus its downstream intergenic region were fused behind the luciferase coding region in a chimeric plasmid for transient transfections, luciferase activity increased 7-fold in amastigotes and decreased 5-fold in epimastigotes. Thus, developmental expression of these alternating genes is regulated by different mechanisms.
Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, constitutes a major public health problem throughout much of Latin America. During its life cycle in the reduviid bug vector and a mammalian host, the parasite goes through two extracellular stages called the epimastigote and trypomastigote forms and one intracellular stage called the amastigote form. In mammalian hosts, the parasites exist mainly as intracellular amastigotes in a variety of cell types. Relatively little is known about the biochemical and immunological properties of this intracellular form compared with the epimastigote form, primarily for the practical reason that epimastigotes grow readily in cell-free culture, whereas amastigotes can only be obtained from infected animals or cultures of mammalian cell lines.
Recently, we showed that the amastigote form of T. cruzi has on its surface a family of closely related glycoproteins collectively called amastin whose mRNA level is at least 50-fold higher in amastigotes than in epimastigotes and trypomastigotes(1) . The 174-amino acid sequence of nascent amastin has four distinct hydrophobic domains of 20-30 amino acids each, suggesting that the protein may span the outer membrane. The biological function of amastin is not known, but its abundance and surface location suggest that it plays an important role in the interaction between amastigotes and their cytoplasmic environment.
Amastin is encoded by eight or
more tandem genes of 1.2 kb ()each that are located within
3-kb DNA repeats. These genes are constitutively transcribed at the
same rate in all developmental stages, indicating that their elevated
expression in amastigotes must be regulated
post-transcriptionally(1) . Many tandem genes in T. cruzi and other trypanosomatids, such as Leishmania species and
African trypanosomes, are known to be transcribed into polycistronic
precursor
RNAs(2, 3, 4, 5, 6, 7) .
These precursor transcripts are processed into individual mRNAs by
intergenic cleavages followed by addition of a 39-nucleotide spliced
leader (SL) to the 5` ends in a trans-splicing reaction and
addition of a poly(A) to the 3` ends via
polyadenylation(8, 9, 10) . Thus, if the
amastin gene cluster is transcribed polycistronically, the
post-transcriptional control of amastin mRNA abundance is likely to be
regulated at these processing steps or by stability conferred on the
mRNA by the 5`- or 3`-untranslated regions (UTRs).
Another unusual
feature of gene expression in trypanosomatids is the apparent lack of
promoters at the beginning of tandem gene clusters where transcription
by RNA polymerase II could be initiated. To date, the only identified
promoters for protein-encoding genes of trypanosomatids are in African
trypanosomes. In these organisms, the promoters for genes encoding two
different surface proteins, the variant surface glycoprotein (VSG) and
the procyclic acidic repetitive protein (PARP), were detected by their
ability to drive expression of a reporter gene in transfected
cells(11, 12) . However, these genes appear to
represent a special case because their transcription is resistant to
-amanitin, suggesting that similar to rRNA genes, they are
transcribed by RNA polymerase I or a modified form of RNA polymerase
II(13, 14) . In contrast, transcription of the amastin
genes of T. cruzi and the other protein-encoding genes of
trypanosomatids examined to date is inhibited by
-amanitin,
indicating that they are transcribed by a conventional RNA polymerase
II.
Two reports describing expression of foreign genes in T. cruzi indicated that the presence of properly oriented 5` sequences is essential for expression of a reporter gene. In one study a segment of the T. cruzi SL gene inserted in front of chloramphenicol acetyltransferase gene was shown to facilitate expression of chloramphenicol acetyltransferase(15) , and in another report, expression of the neomycin phosphotransferase and chloramphenicol acetyltransferase genes was obtained following transfection with a plasmid bearing these genes flanked by regions of the glyceraldehyde 3-phosphate dehydrogenase gene(16) . Since neither of these studies defined the sequences required for mRNA expression in T. cruzi, we undertook the present study to search for potential regulatory elements upstream of or within the amastin gene family. We discovered that the 3-kb repeats contain a previously unknown gene of 1.5 kb in addition to the 1.2-kb amastin gene, leaving intergenic regions (IRs) of only 111 and 145 bp between these two gene classes. The new gene potentially encodes a protein we have named tuzin for T. cruzi expressed protein. Since the steady-state levels of both amastin mRNA and tuzin mRNA vary dramatically among the three developmental stages, the signals for their respective post-transcriptional regulation likely occur within their UTRs or short IRs. We show here that indeed the 3`-UTRs and IRs contribute to the differential expression of amastin and tuzin mRNAs in the different developmental stages.
To generate plasmid pL3T containing the 5`-flanking
region of the amastin cluster, a 2.9-kb HindIII-PstI
fragment located upstream the first amastin gene in recombinant phage
-A11 was used to replace a HindIII fragment containing
the intergenic region in plasmid pLIT. Plasmid pL2T was prepared by
replacing the same segment in pLIT with a 3-kb HindIII
fragment in
-A11 containing both the amastin and tuzin genes.
Plasmids containing the T. cruzi rRNA gene promoter (pLRT)
were generated from plasmid pLST by inserting a 580-bp HincII T. cruzi rRNA gene spacer fragment into the unique HindIII site. This fragment was isolated from plasmid
pTc18S(24, 25) . Plasmids pLRA and pLRC are
derivatives of pLRT in which the TCR27 3`-UTR was replaced by fragments
corresponding to the 3`-UTR and polyadenylation sites of an amastin
gene and a tuzin gene, respectively. The amastin 3`-UTR fragment was
obtained by digesting plasmid pAB3 with SphI and MscI, generating an 860-bp fragment containing the complete
3`-UTR plus a 200-bp segment spanning the polyadenylation site and SL
addition site of the downstream tuzin gene. The tuzin 3`-UTR was
obtained by PCR amplification of plasmid pAB3 using primers
corresponding to the 5` end of the amastin coding region (P1) and the
3` end of the tuzin gene (P3). Primers P1 (see above) and P3
(5`-TGCTGTCGACAGTAGCCAGCCATG) contain ApaI and SalI
sites, respectively, to facilitate cloning. Finally, plasmid pHDT was
constructed by deleting the 360-bp BamHI-PstI
fragment containing the PARP 3`-UTR in the pHD1 plasmid (26) and inserting the 520-bp HincII-StuI
fragment containing the 3`-UTR of the TCR27 gene.
Figure 1:
Diagram of the 5` region of the
amastin/tuzin gene cluster and the sequence of an interior tuzin gene
plus its flanking regions. A,large and smallshadedrectangles indicate the amastin coding
regions and 3`-UTRs, respectively. Openrectangles represent the tuzin genes whose 3`-UTRs of 0-20 bp are too
small to show here. Restriction sites are for BamHI (B), BglII (Bl), EcoRI (E), PstI (P), and SphI (S). The openarrowhead indicates the
position at which the sequence preceding the entire gene cluster
diverges from the sequence preceding the interior tuzin genes, and
corresponds to the openarrowhead in panelB. The genomic DNA segment in bacteriophage clone
-A11 is denoted by the hatchedline. Three
fragments derived from either the 5` end or interior regions of the
gene cluster that were used in the transfection experiments shown in Fig. 4are denoted by the patternedboxes. Lines labeled 1-5 indicate fragments
that were probed in the nuclear run on experiment shown in Fig. 3. B, the sequence of a 2411-bp region between the
coding regions of adjacent amastin genes with the amastin stop and
start codons at the 5` and 3` ends, respectively. The start and stop
codons of the intervening tuzin gene are denoted by the underlyingblackbox and blackoval,
respectively. BoxedAG dinucleotides indicate SL
addition sites. Polypyrimidine sites upstream of these SL sites are
also boxed. Blackarrowheads indicate
poly(A) addition sites. The openarrowhead between
the SL addition site and start codon of the tuzin gene indicates the
location at which the unique sequence upstream of the gene cluster
diverges from the intergenic sequence. The indicated restriction sites
were used to construct the plasmids shown in Fig. 4and Fig. 5. C, the deduced amino acid sequence of
tuzin.
Figure 4: Identification of sequences preceding or within the amastin/tuzin gene cluster that facilitate expression of a luciferase reporter gene in epimastigotes. Restriction fragments indicated by the patternedboxes in Fig. 1A were cloned in either forward or reverse orientation at the 5` end of the luciferase gene in the pGEM-luc vector. The smallblackboxes indicate the locations of a 73-bp fragment containing the amastin SL addition site and its upstream polypyrimidine tract. A 520-bp fragment containing the 3`-UTR of the TCR27 gene and extending 135 bp beyond its polyadenylation site was cloned downstream of the luciferase gene. T. cruzi epimastigotes were transfected with 100 µg of each of the depicted plasmids and assayed for luciferase activity 48 h later.
Figure 3:
Analysis of transcripts from the
5`-flanking region of the amastin/tuzin gene cluster. A,
photograph of an ethidium bromide-stained agarose gel containing the
cloned restriction fragments 1-5 shown in Fig. 1A (1-5), pBluescript (v), a fragment
encoding part of the 24 S rRNA (r) and two fragments of
the T. cruzi TCR27 gene (T). B,
autoradiogram of labeled run on transcripts hybridized to the DNA
fragments shown in panelA. Migrations of molecular
size markers in kb are shown on the left.
Figure 5: Effect of different 3`-UTRs and downstream IRs on luciferase expression in epimastigotes and amastigotes of T. cruzi. Plasmid pLRT contains the promoter for a T. cruzi rRNA gene (24) inserted in the forward orientation at the HindIII site upstream of the amastin SL site in plasmid pLST. In the pLRA and pLRC plasmids, the TCR27 3`-UTR of pLRT was replaced by the amastin 3`-UTR plus downstream IR, and by the tuzin 3`-UTR plus downstream IR, respectively. T. cruzi epimastigotes and amastigotes were transfected with 20 and 100 µg, respectively, of each plasmid and luciferase assays were performed 48 h later. In the Foldincrease columns, the numbers in brackets were obtained by normalizing the luciferase activity units of pLRT to 1 and then expressing the luciferase activity units of pLRA and pLRC relative to that normalized value. For example, in amastigotes pLRA expresses 7.2-fold more luciferase than pLRT. The ratio of these normalized values in amastigote transfectants versus epimastigote transfectants is shown in the last column.
In addition to the above characterization, 3-kb fragments from BglII- or EcoRI-digested T. cruzi DNAs were eluted from an agarose gel and ligated into pBluescript. After colony hybridization with an amastin cDNA, two plasmids with 3-kb inserts, named pAB3 and pAE3, were chosen for study. In both inserts, the sequence between the stop codon of the upstream amastin gene and the start codon of the downstream amastin gene was determined, and this segment of the pAE3 insert is shown in Fig. 1B. This intergenic sequence was compared with the sequence upstream of the first amastin gene in the cluster. The two sequences are completely different upstream of the openarrowhead shown in Fig. 1, A and B, and nearly identical downstream of this position. In the intergenic sequence shown in Fig. 1B, distinctive polypyrimidine tracts occur just upstream of the point of divergence. Polypyrimidine tracts have been found to be essential for trans-splicing of the SL in Trypanosoma brucei and Leishmania enrietti(29, 30) , and this motif provided the first hint that the 3-kb repeats might contain another gene besides the amastin gene.
To determine if the tuzin region is transcribed, we
prepared Northern blots of RNAs isolated from the epimastigote,
amastigote, and trypomastigote stages. The blot shown in Fig. 2was probed in succession with the coding regions for tuzin (panelB), amastin (panelA), and
24 S rRNA (31) (panelC). The amastin
and tuzin RNAs are 1.4 and 1.7 kb, respectively, indicating from a
comparison with their respective gene lengths that each has a 3`
poly(A) tail of about 200 nucleotides. A densitometer tracing of a
short exposure of the autoradiogram shown in panelA indicated that the ratio of amastin RNA in epimastigotes,
amastigotes, and trypomastigotes is 1:85:4.8, respectively. The
corresponding ratio for the tuzin RNA was 1:3.5:0.1, whereas that for
rRNA was 1:1:1, demonstrating that equal amounts of total RNA were
added to each lane. In addition, the tuzin autoradiogram shown in Fig. 2was exposed about 10 times longer than the amastin
autoradiogram, indicating that in the amastigote and trypomastigote
stages amastin RNA is much more abundant than tuzin RNA. Our earlier
measurements suggested that amastin RNA is about 50-fold more abundant
in amastigotes than in epimastigotes(1) , but the difference
between that value and the 85-fold found here could reflect differences
in the growth phases of the different developmental stages when the RNA
was collected. Thus, it is likely that the two alternating gene classes
are transcribed into large polycistronic precursor RNAs, which are
processed into amastin and tuzin mRNAs whose steady state levels are
quite different in the amastigote and trypomastigote stages but much
more similar in the epimastigote stage. Since the specific activities
of the radioactive amastin and tuzin gene probes used in panelsA and B may be different, we cannot use the
Northern blots to quantitate the relative abundances of amastin and
tuzin mRNAs in epimastigotes. However, about the same number of amastin
and tuzin cDNA clones were detected in a T. cruzi epimastigote
cDNA library, suggesting that the levels of their corresponding mRNAs
are similar at this developmental stage.
Figure 2:
Northern blot analysis of transcripts from
the amastin/tuzin gene cluster. The blot containing total RNAs from
epimastigotes (E), amastigotes (A), and
trypomastigotes (T) was probed in succession with a
PCR-generated fragment from the tuzin coding region (panel B,
positions 1131-1648 in Fig. 1B), an amastin cDNA (panelA), and a fragment containing part of the 24
S rRNA gene (panelC). Exposure times of the
autoradiograms were 4.5 h (A), 48 h (B), and 15 min (C). In panelB, the blackspot just above the band in the Tlane is an imperfection in the x-ray film and not due to probe
hybridization.
To map the 5` and 3` ends of the tuzin mRNA, five tuzin cDNAs were isolated from about 10,000 clones of an amastigote cDNA library. The same number of library clones yielded more than 100 amastin cDNA clones, consistent with the Northern blot that suggested amastin mRNAs are about 20 times more abundant in amastigotes than tuzin mRNAs. Partial sequencing of the five tuzin cDNA clones demonstrated that in four cases the polyadenylation sites were located either seven or 20 nucleotides beyond the stop codon, and in one case polyadenylation occurred at the A residue within the UAG stop codon itself, as indicated in Fig. 1B. The RNA molecule that gave rise to this latter cDNA apparently did not have any 3`-UTR nucleotides other than the poly(A), but its translation termination was preserved because the UAG was converted to a UAA stop codon.
Since none of the five tuzin cDNAs contained a 5` SL, PCR amplification was performed on an sample of the cDNA library using a SL primer and a primer complementary to nucleotides 886-906 in the tuzin coding region. The sequence of the resulting 140-bp amplification product demonstrated that the AG dinucelotide at position 777 serves as the main splice acceptor site for the SL (indicated in Fig. 1B). This dinucleotide is the first AG downstream of the previously mentioned polypyrimidine tract. Five additional AG dinucelotides occur between this site and the start codon, so some tuzin mRNAs might have alternative SL addition sites as is the case with amastin mRNAs(1) .
No luciferase activity above background was detected when unmodified pGEM-luc or pLT, a derivative containing the TCR27 3`-UTR, were introduced. However, when a 73-bp segment containing the polypyrimidine tracts and SL addition site immediately preceding the amastin coding region was placed in front of the luciferase gene of pLT, a 400-fold increase in luciferase activity over the background level was detected (pLST). This result provides strong evidence that a SL addition site is necessary for the production of luciferase mRNA, and this 73-bp segment was included in all subsequent plasmid constructs.
The three plasmids shown in the middle of Fig. 4(pL3T, pL2T, and pLIT) each contain a different region of the amastin/tuzin gene cluster cloned in front of the luciferase gene and the 73-bp segment. Thus, if any of these three regions contains promoter activity, the amount of luciferase detected when its plasmid is introduced into T. cruzi epimastigotes should increase relative to that of pLST. As indicated in Fig. 1, the cloned region in pL3T includes the first tuzin gene in the gene cluster plus about 1.5 kb of unique sequence preceding this gene. The region in pL2T is a complete internal 3-kb repeat including an amastin gene, a tuzin gene, and their downstream IRs. The third region, cloned in pLIT, contains only a tuzin gene and its 5`- and 3`-flanking intergenic regions. For each of these constructs, the amount of luciferase activity is about 700-fold above background (range = 692-747-fold) compared with 400-fold above background for pLST. This 1.75-fold increase in luciferase activity might be due to a small amount of promoter activity or due to the presence of additional sequences upstream of the 73-bp segment that also influence trans-splicing. In either event, no region that clearly contains substantial promoter activity was detected (see Fig. 5for an example of a T. cruzi DNA sequence that does have promoter activity). In particular, note that in pL3T the 1.5-kb region immediately upstream of the gene cluster does not have promoter activity. Regions still further upstream were not investigated because the nuclear run on experiments (Fig. 3) indicated that these regions, encompassing fragments 1 and 2 shown Fig. 1, were not transcribed. When the orientation of these three fragments was reversed, the luciferase activity was about the same as that for pLST (not shown).
The only trypanosomatid in which promoters for
protein-encoding genes have been identified is the African trypanosome, T. brucei, and in these cases the promoters have been shown to
be resistant to -amanitin, which is a characteristic of RNA
polymerase I promoters (11, 12, 13, 14) . Since our results
suggest that the signals for SL addition alone are sufficient for
luciferase activity in transfected epimastigote cells, we tested
whether the promoter for one such
-amanitin resistant gene of
African trypanosomes, the PARP gene(12, 26) , would
stimulate luciferase expression in T. cruzi. As shown at the
bottom of Fig. 4, plasmid pHDT has a 290-bp fragment containing
the PARP promoter and SL addition site (26) inserted upstream
of the luciferase gene, and the 3`-UTR of the TCR27 gene inserted
downstream. This plasmid directs about 900-fold more luciferase
activity than background, or only 1.3-fold (900- versus 700-fold) more than pLIT, pL2T, or pL3T, which contain regions
derived from the amastin/tuzin gene cluster. Although it is not
possible to interpret unambiguously the result of this heterologous
PARP transfection, it does suggest that the 290-bp fragment of T.
brucei contributes functional SL addition signals for the
luciferase gene but little, if any, promoter activity in T.
cruzi. Thus, a promoter for a protein-encoding gene of African
trypanosomes is not recognized as a promoter by epimastigotes of T.
cruzi.
Unfortunately, repeated attempts to transiently transfect amastigote cells under a variety of electroporation conditions with plasmids pLIT and pLST shown in Fig. 4, or with corresponding plasmids containing the amastin 3`-UTR + IR, did not detect any luciferase activity above background (not shown). These negative results suggest that electroporation is much less efficient for transfection of amastigotes than for epimastigotes. Therefore, we had to construct an improved vector that directed much higher levels of luciferase activity. The new construct was based on the observation that in African trypanosomes high expression levels of a reporter gene are only achieved using promoters for genes encoding rRNA, PARP, and the VSG, each of which is a promoter for RNA polymerase I or a RNA polymerase I-like enzyme. Since the promoter for a T. cruzi rRNA gene has recently been reported(24, 25) , we inserted a 580-bp fragment containing this promoter in front of the amastin SL addition site of pLST and obtained the results shown in Fig. 5. The presence of this T. cruzi rRNA promoter increased the amount of luciferase activity in transfected epimastigotes so dramatically that we reduced the amount of plasmid in a typical transfection from 100 to 20 µg and conducted the luciferase assays with a 1:50 dilution of the cell extracts. Thus, the results with pLST shown in Fig. 5are not directly comparable with the results shown in Fig. 4because of the differing transfection and assay conditions.
When transfected into epimastigotes, pLRT containing the T. cruzi rRNA promoter and the 3`-UTR of the TCR27 gene
stimulated about 2,050 times more luciferase activity (271,098- versus 132-fold) than did the corresponding plasmid without
the promoter (pLST) or with the promoter in the opposite orientation.
When transfected into amastigotes, pLRT containing the correctly
oriented promoter resulted in only a 94-fold increase in luciferase
activity rather than the 2,050-fold increase seen in epimastigotes,
consistent with the prediction that transfection efficiency is much
lower in amastigotes than in epimastigotes. However, this value in
amastigotes still represents a substantial number of luciferase units
above background (15,000 versus about 170), so plasmids in
which luciferase expression is driven by the rRNA gene promoter were
used to compare the effects of different 3`-UTRs + IRs. As shown
in Fig. 5, in epimastigotes the 3`-UTR of the constitutively
expressed TCR27 gene (pLRT) conferred the highest level of luciferase
expression, whereas the presence of the amastin 3`-UTR + IR (pLRA)
resulted in a 5-fold drop in luciferase expression. The opposite effect
was seen in amastigote cells. Luciferase activity was 7.2-fold higher
with the amastin 3`-UTR + IR than with the TCR27 3`-UTR. As
indicated in the right-hand column of Fig. 5, the resulting
normalized difference between amastigotes and epimastigotes is 36-fold
(5 7.2), which is not much less than the 50-85-fold
difference in the steady-state level of amastin mRNA observed on
Northern blots (see Fig. 2). Likewise, for the tuzin 3`-UTR
+ IR, the normalized ratio in luciferase activity between
amastigotes and epimastigotes is 3, consistent with the Northern blot
in Fig. 2, indicating that there is 3.5 times more tuzin mRNA in
amastigotes than epimastigotes.
Furthermore, in amastigotes, the presence of the tuzin 3`-UTR + IR (pLRC) had the opposite effect of the amastin 3`-UTR + IR on luciferase activity. When transfected into amastigotes, the tuzin region caused the luciferase activity to drop to 0.3 of that expressed with the TCR27 3`-UTR, whereas the amastin 3`-UTR + IR increased it by 7.2-fold. In contrast, in epimastigotes the tuzin and amastin 3`-UTRs + IRs had similar affects. Compared with the TCR27 3`-UTR, the tuzin 3`-UTR + IR caused a 10-fold drop, and the corresponding amastin region caused a 5-fold drop. Thus, the tuzin 3`-UTR + IR resulted in a lower level of the chimeric luciferase mRNA in both epimastigotes and amastigotes, whereas the amastin 3`-UTR + IR caused an increase in amastigotes and a decrease in epimastigotes.
T. cruzi epimastigotes and amastigotes were incubated in the presence of either 200 or 500 ng/ml cycloheximide for 4 h and total RNA isolated for Northern blots that were probed in succession with the coding regions of tuzin, amastin, and rRNA (Fig. 6). Again, trypomastigotes were not used because of the difficulty in obtaining a sufficient number for this experiment. Densitometric measurements of the rRNA signals (panelC) were used to adjust for differences in RNA loaded in each lane. After this normalization, the relative signal intensities in panelsA and B were determined and several conclusions were drawn. First, the two different cycloheximide concentrations had very similar effects in all cases, so the RNA levels in the two concentrations could be averaged. Second, the tuzin autoradiogram shown in Fig. 6was exposed about 10 times longer than the amastin autoradiogram, again reflecting the lower abundance of tuzin mRNA. Third, in amastigotes the effect of cycloheximide on amastin RNA and tuzin RNA was quite different. At this developmental stage, the drug caused a 3-fold drop in amastin RNA and a 7-fold increase tuzin RNA. The magnitude of this 7-fold increase is not readily apparent in panelB because of the adjustment that must be made for more RNA in the 0 drug lane of amastigotes as shown in panelC. Fourth, in epimastigotes the presence of cycloheximide did not affect substantively the steady-state level of amastin mRNA (i.e. 1.3-fold more with cycloheximide), whereas the tuzin mRNA level increased by 4.3. Finally, no differences were observed between 4 h of incubation in cycloheximide (Fig. 6) and 2 or 8 h of incubation (not shown).
Figure 6:
Effect of cycloheximide on the
steady-state levels of amastin and tuzin mRNAs. For these Northern
blots, total RNAs were extracted from epimastigotes (E) and
amastigotes (A) that had been incubated for 4 h in the
presence of 0, 200, or 500 ng of cycloheximide/ml. Filters were probed
in succession with the coding regions for tuzin (panelB), amastin (panelA), and 24
rRNA (panelC). The signal intensities on short
exposures of the autoradiograms were determined by densitometry. The
rRNA signals in each lane of panelC were normalized
to each other, and the relative signals of the amastin and tuzin mRNAs
were adjusted to this normalized value. Exposure time for the tuzin
autoradiogram (panelB) was about 10 times longer
than for the amastin autoradiogram (panelA).
The interpretation of these cycloheximide results is tempered by several factors (see ``Discussion''), but one clear observation is that in amastigotes the inhibition of protein synthesis causes a decrease in amastin mRNA and an increase in tuzin mRNA. In contrast, in epimastigotes, this inhibition has no effect on amastin mRNA but causes an increase in tuzin mRNA similar to that seen in amastigotes (4.3-fold increase versus 7-fold increase). Thus, translation inhibition affects the two mRNA species differently.
The results described here suggest that different molecular mechanisms regulate the RNA levels of the alternating amastin and tuzin genes in the three developmental stages of T. cruzi. The simplest model consistent with our results and with previous findings in other trypanosomatids (8, 34, 39) is that the two alternating genes are constitutively transcribed into large polycistronic precursor RNAs, which are processed into amastin and tuzin mRNAs whose levels are post-transcriptionally regulated by different mechanisms. The first gene in the cluster, a tuzin gene, does not contain the upstream polypyrimidine tract and AG acceptor site for SL addition that precede internal tuzin genes (see Fig. 1B), suggesting that this first gene is not processed into a functional mRNA. No evidence was obtained for the presence of a strong promoter either preceding or within the amastin/tuzin gene cluster. One of several possible explanations for this lack of a promoter is that polycistronic transcription of the gene cluster is initiated weakly at a number of sites within the cluster and that the overall chromatin configuration surrounding the cluster imparts the signals for the unidirectional transcription.
Regions
that likely participate in the different post-transcriptional
regulatory mechanisms of these two coding regions are their flanking
5`- and 3`-UTRs and their flanking IRs. Consistent with this
possibility are reports from other experimental systems that 3`-UTRs
are involved in phenomena as diverse as self-regulation of mRNA
degradation(40, 41) , rates of
translation(42) , mRNA localization (43) , cellular
growth and differentiation(44) , and tumor suppression (45) . Recently, a stem-loop structure in the 3`-UTR of the
PARP gene of African trypanosomes was reported to participate in its
differential regulation(46) . In addition, sequences of both
the 3`- and 5`-UTR of hsp83 mRNA in Leishmania were found to
be involved in its temperature-dependent regulation(47) . The
IR sequences downstream of the 3`-UTRs also have been found to be
important. This region contributes to the differential expression of
the three gene classes encoding the surface protease gp63 of Leishmania chagasi, whereas the 3`-UTRs by themselves have
little effect (34) . Likewise, within the IR following the
dihydrofolate reductase-thymidylate synthase gene of Leishmania
major, and between the - and
-tubulin genes of T.
brucei, the SL addition site of the downstream gene directs the
location of the upstream gene's poly(A) addition
site(8, 48) .
Amastin mRNA has a short 5`-UTR (17 nt) and a long 3`-UTR (630 nt), whereas tuzin mRNA has a long 5`-UTR (137-nucleotide) and a short (<20-nucleotide) or nonexistent 3`-UTR. None of these UTRs nor the intervening IRs of 145 and 111 bp display any obvious sequence similarities, consistent with the possibility that these sequences participate in the differential expression of amastin and tuzin mRNAs. In the case of tuzin mRNA, its short or nonexistent 3`-UTR suggests that this sequence does not contribute much to the differential regulation of tuzin mRNA. The complete absence of a 3`-UTR in one tuzin cDNA and the creation of its UAA stop codon by 3` polyadenylation is reminiscent of a similar phenomenon in mammalian mitochondrial DNA in which the termination codons in the mRNAs of 9 of the 13 protein-encoding genes are created during poly(A) addition (49) .
Two general models have been proposed to account for
those cases in other biological systems where cycloheximide affects the
level of an mRNA in a post-transcriptional
manner(42, 50) . One model invokes the existence of a
labile regulatory protein that either stabilizes a specific mRNA or
targets it for degradation(50) . The other model, shown for
mammalian -tubulin mRNA, involves the co-translational degradation
of mRNAs by a ribosome-associated RNase(51, 52) . In T. cruzi amastigotes, cycloheximide caused a decrease in the
abundant amastin mRNA and an increase in the less abundant tuzin mRNA,
consistent with the presence of both the positive and negative
regulatory aspects of the first model. In epimastigotes, the
cycloheximide had no effect on amastin mRNA and caused an increase in
tuzin mRNA similar to that observed in amastigotes. This result
suggests that the cycloheximide-affected mechanism regulating tuzin
mRNA levels may be the same in amastigotes and epimastigotes, whereas
the cycloheximide-affected mechanism contributing to the elevated
amastin mRNA level in amastigotes does not operate in epimastigotes.
More experiments will be required to elucidate the details of these
cycloheximide-affected mechanisms, including measurements of the mRNA
decay rates and the use of other translation inhibitors such as
pactamycin or puromycin that act on different steps in protein
synthesis than does cycloheximide(53) .
Transient transfections of the luciferase reporter gene containing the different 3`-UTRs + IRs also indicated that different regulatory mechanisms control the amastin and tuzin mRNA levels (Fig. 5). From these experiments, we cannot determine whether it is the 3`-UTRs or the IRs, or both, that are responsible for this difference. However, when similar reporter gene transfections were conducted in Leishmania using the different 3`-UTRs and the downstream IRs of the three gp63 gene classes of L. chagasi, it was found that the 3`-UTRs and the IRs each contribute to the differential expression of the three gene classes(34) . By analogy, it seems likely that a similar scenario will hold for the differential expression of the amastin/tuzin gene cluster.
Finally, the deduced amino acid sequence of tuzin (Fig. 1C) does not have any substantive similarities with the protein sequences currently in the data bases. In contrast to amastin, which is an extremely hydrophobic protein, tuzin is a highly charged molecule lacking a signal peptide or other sequence motifs that might provide insight into its cellular location and biological function. The relatively low abundance of its mRNA suggests that tuzin is a rare protein that will be difficult to detect in Western blots of subcellular fractions or in cellular immunolocalization assays. It also is difficult to say whether the alternating arrangement of the amastin and tuzin genes means that their gene products are associated physiologically or structurally. Other known co-transcribed, multi-gene clusters of trypanosomatids encode proteins such as ubiquitin and calmodulin(6) , a phosphatase, and an RNA polymerase subunit(7) , and the VSG, a transferrin receptor and adenylate cyclase(54) . It is possible that these gene products are associated, i.e. the phosphatase could act on the RNA polymerase, or the transferrin and adenylate cyclase might be attached to the same membrane as the VSG, but in no case has such a relationship been demonstrated. Thus, the reason for the alternating nature of the amastin and tuzin genes remains unclear.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U25030[GenBank].