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The 3[prime] untranslated region of the hsp 70 genes maintains the level of steady state mRNA in Trypanosoma brucei upon heat shock
Introduction
Materials And Methods
Trypanosomes
Stable DNA transformation
Genomic Southern blot analysis
Pulsed field gel electrophoresis (PFGE)
RNA preparation and northern analysis
Reverse transcription and PCR
Description of DNA fragments used in plasmid constructs
Results
Temperature influence on mRNA levels
Generation of stable transformants expressing different versions of the hygromycin phosphotransferase reporter gene
Temperature effects on the steady mRNA levels in different transformed cell lines
Determination of the 3[prime] ends of hph mRNAs
Discussion
Acknowledgements
References
The 3[prime] untranslated region of the hsp 70 genes maintains the level of steady state mRNA in Trypanosoma brucei upon heat shock
ABSTRACT
INTRODUCTION
African trypanosomes are unicellular eukaryotic flagellates which cause sleeping sickness in man and related diseases in livestock. They have an intricate life cycle which alternates between a mammalian host and the transmitting insect vector, the tsetse fly (1,2). When parasites are transmitted from the tsetse fly into the mammalian host, the parasites experience a rapid temperature elevation from ~25 to 37°C. During this process, parasites differentiate from the insect adapted procyclic form (or insect form) to the bloodstream form, and extensive changes in cell morphology and metabolism occur. The exposure to elevated temperatures also results in an increased expression of the heat shock 70 kDa protein (hsp 70) gene in the bloodstream form Trypanosoma brucei, compared to that in the procyclic form (3-5). Temperature shifts may serve additional roles in protozoa which have life cycles in organisms with different ambient body temperatures. For instance, a temperature shift is sufficient to induce differentiation of the related protozoan Leishmania (5-8). The adaptation of these organisms to survival over a wide temperature range suggests that heat shock responses may play an important role in parasite survival and differentiation.
The heat shock response is an evolutionarily conserved reaction. Virtually all organisms analyzed to date increase the synthesis of a set of conserved hsps in response to environmental stress (9). Among these hsps, hsp 70 proteins are most conserved. Some of the hsps are constitutively expressed, while others increase their level of expression upon a temperature shift. The induction of hsp gene expression by elevated temperatures is primarily regulated at transcriptional levels in most eukaryotes. The temperature sensitive transcription results from the binding of heat shock transcription factors to the conserved 14 nt sequences of a heat shock transcription factor binding element in the hsp gene promoters, leading to the activation of hsp genes (9-13). However, unlike other eukaryotes, transcriptional induction by heat shock does not occur in T.brucei (14). This property indicated that the control mechanism for hsp 70 genes expression in T.brucei does not follow the conserved transcriptionally mediated mechanism described for other eukaryotes (14,15).
Transcription of protein coding genes and mRNA maturation in trypanosomes and other kinetoplastida involves mechanisms which are different from those in most higher eukaryotes (16,17). Every mRNA in trypanosomes contains two exons, the 5[prime] mini-exon and the main-coding exon, which are transcribed from two separate genes. Through trans-splicing, a capped 39 nt transcript is joined with the main coding exon (16,18,19). Most genes in trypanosomes are organized in tandem arrays and polycistronically transcribed. Through the processing events of poly A addition and trans-splicing, intergenic regions are excised from the precursor RNA resulting in the generation of the mature mRNAs. Polycistronic transcription of genes could occur, either from promoters located upstream of the tandem arrays of genes or from individual promoters located in front of each gene separately. Alternatively, both events could occur simultaneously (as illustrated in Fig.
Figure 1. A diagram of structure and transcription of the hsp 70 locus in T.brucei. Boxes indicated by 1-6 represent hsp 70 genes. Arrows facing down and up indicate the 5[prime] trans-splicing acceptor sites and the 3[prime] end polyadenylation sites, respectively. Lines with arrow heads indicate the location of promoters and the direction of transcription. H23 and H2 are the hsp 70 intergenic region promoter and the 3[prime] end of the hsp 70 gene used in constructs described in the paper. The hsp 70 gene locus of T.brucei contains, from 5[prime] to 3[prime], a cognate hsp 70 gene (gene 1) which is separated by ~5 kb of DNA from a cluster of five identical hsp 70 genes (genes 2-6) (3,4,20; Fig. 
MATERIALS AND METHODS
Trypanosomes
The procyclic trypanosomes used were either T.brucei 427 procyclic stock obtained from Dr R.Brun (427 RB) or the procyclic form derived from T.brucei bloodstream form variant 118 clone 1 by A.Rattray and L.H.T.Van der Ploeg (unpublished; 22). Procyclic trypanosomes were maintained in SDM79medium at 23-25°C as described (23). Procyclic trypanosomes of log phase (4-8 × 106 cells/ml) were used in all the experiments described. For heat shock experiments, aliquots of cultured trypanosomes were transferred into 50 ml tubes and incubated in water baths at different temperatures for different periods of time. Immediately after the heat shock, trypanosomes were spun down and harvested for the analysis of steady state mRNA levels.
Stable DNA transformation
Linearized plasmid DNA (10 or 20 µg) was electroporated into procyclic form trypanosomes using a BTX electroporator as described (24). Thirty-six hours after electroporation, hygromycin B was added to each sample to select for stably transformed trypanosomes. Following limiting dilution cloning, individually transformed trypanosomes were isolated.
Genomic Southern blot analysis
Nuclear DNA was isolated from trypanosomes as described (25). Following the separation of restriction enzyme digested genomic DNA on a 0.8% agarose gel, the DNA was transferred onto nitrocellulose filters. The Southern blots were hybridized with 32P-labeled probes. The final post-hybridizational washes were performed in 0.1 × SSC, 0.1% SDS at 65°C.
Pulsed field gel electrophoresis (PFGE)
The preparation of chromosome sized DNAs and the PFGE analysis were performed as described (26,27). Chromosome size DNAs were separated in a 1% agarose gel for 7 days at 3 V/cm and a pulse frequency of 3300 s. The DNA was transferred onto nitrocellulose filters. The Southern blots were hybridized with 32P-labeled probes. The final post-hybridizational washes were performed in 0.1× SSC, 0.1% SDS at 65°C.
RNA preparation and northern analysis
RNA samples were isolated by GuanSCN lysis and subsequently purified by CsCl centrifugation. Northern analysis of RNAs was carried out by separation of RNA in 1% agarose gels containing 2.2 M formaldehyde. RNA blots were hybridized with 32P-labeled DNA probes. Post-hybridizational washes were performed to a final stringency of 0.1× SSC, 0.1% SDS at 65°C.
Reverse transcription and PCR
The 3[prime] ends of hph mRNAs were mapped using a reverse transcriptase PCR procedure (RT-PCR). The primer XhoT12 (5[prime]-CCTCGAGTTTTTTTTTTTT-3[prime]) was used for reverse transcription. The resulting cDNAs were first amplified with an hph specific primer (primer hph-423: 5[prime]-CGGTCTAATACACTACATGGC-3[prime]) and an XhoT12 primer. Amplified DNAs were subjected to a second PCR reaction using the XhoT12 primer and a second hph specific primer (Primer hph-701: 5[prime]-AACATCTTCTTCTGAAGGCCG-3[prime]) which located downstream of the primer hph-423. Amplified products were subcloned into M13 vector for nucleotide sequence analysis.
Description of DNA fragments used in plasmid constructs
The PARP promoter fragment was as described (24). The hsp 70 intergenic region promoter fragment (H23) consists of sequences located upstream of the ATG of the hsp 70 gene 3 to the HindIII site located at the 3[prime] end of the coding region of the hsp 70 gene 2, as described (21). The [beta][alpha] tubulin intergenic region sequence (T) extends from the EcoRI site located at the 3[prime] end of the [beta]-tubulin gene to the immediate upstream of the ATG of the [alpha]-tubulin gene (28). The 3[prime] end DNA fragment of hsp 70 gene 2 (H2) is of a size of 432 bp and extends from 21 bp upstream of the TAA codon of the hsp 70 gene 2 to the MluI site located 11 bp upstream of the major 3[prime] splice acceptor site (AG) of the hsp 70 gene 3 (20).
RESULTS
Temperature influence on mRNA levels
Procyclic trypanosomes are normally maintained at 25°C. It was previously shown that in procyclic trypanosomes (strain 427 RB), the hsp 70 mRNA level increased when the incubation temperature was raised to 41-42°C over a period of 180 min (3). Since the procyclic trypanosome 427 RB has been maintained in laboratories for >15 years, it was questioned whether the up-regulated expression of hsp 70 genes upon heat shock is generally observed among distinct procyclic trypanosome isolates. Therefore, the temperature influence on the steady state mRNA level in response to temperature shifts was analyzed in a recently established procyclic trypanosome derived from bloodstream form variant 118 clone 1. The procyclic 118 clone 1 trypanosome used in heat shock experiments was maintained in culture for <6 months. Incubation of the procyclic 118 clone 1 at 37, 39 and 41°C, respectively, for different periods of time, up to 180 min, did not lead to a significant induction of the hsp 70 gene expression, i.e. the level of hsp 70 mRNA remained at an equally high or a slightly increased level, compared to that of normally grown (at 25°C) procyclic trypanosomes (Fig.
Figure 2. The steady state mRNA level of protein coding genes in response to elevated temperatures. The RNA samples were isolated from procyclic 118 clone 1 trypanosomes that were maintained at 41°C for various periods of time. Lane 1 represents total RNA from trypanosomes maintained at a normal growing temperature of ~25°C. Lanes 2, 3 and 4 represent total RNAs derived from trypanosomes incubated at 41°C for 30, 60 and 180 min, respectively. RNA samples were separated on a 1% formaldehyde-agarose gel and transferred to nitrocellulose filters. The filter was sequentially hybridized with different 32P-labeled probes as indicated on the top of each panel. Signals on the filters were stripped off prior to hybridization with a different probe. Following hybridization, filters were washed to a final stringency of 65°C, 0.1× SSC and 0.1% SDS. The small box under the Hsp panel represents the hybridization with the 18 S rRNA gene probe (30), demonstrating that equal amounts of total RNA were loaded in each lane. Abbreviations and hybridization probes: Hsp, the 1.8 kb HindIII fragment derived from the hsp 70 gene coding region (20); Tub, the HindIII-EcoRI fragment of the [beta]-tubulin coding region (28); CRAM, the CRAM cDNA clone (29); PARP, the cDNA clone cPT4 (31). I further examined the effect of temperature on the expression of other protein coding genes. Surprisingly a heat shock at 39 or 41°C drastically reduced the steady state mRNA levels for many other genes in procyclic trypanosomes (Fig. By nuclear run-on analysis, I have previously shown that heat shock at 41°C for 25 min reduced the transcriptional efficiency of the RNA pol II transcribed hsp 70 and tubulin genes to ~70-50% of their original transcriptional efficiency and reduced the pol I transcribed rRNA gene and the PARP gene to ~10% of their original transcriptional efficiency (14). Heat shock of procyclic trypanosomes for 120 min at 41°C abolished the transcription of almost all genes including the hsp 70 genes (14). Thus, to a limited extent, the reduction of transcriptional efficiency upon heat shock may have resulted in the decreased steady state mRNA levels of some genes. However, the change of transcriptional efficiency does not completely explain the change of the relative amount of steady state mRNA levels. Other mechanisms affecting the turnover rate of mRNA encoded by different genes upon heat shock are most likely involved. I further searched for unique sequences in the hsp 70 mRNA that may affect mRNA abundance during a heat shock.
Generation of stable transformants expressing different versions of the hygromycin phosphotransferase reporter gene
To determine sequences affecting the abundance of mRNA upon elevated temperatures, I investigated the temperature effect on the level of mRNA of the hygromycin phosphotransferase (hph) reporter gene driven by different promoters and flanked by the 5[prime] untranslated region (UTR) or 3[prime] UTR from different genes. The extent of heat shock affecting mRNA driven by the PARP promoter and its 3[prime] splice acceptor site was compared to that driven by the hsp 70 intergenic region promoter and its 3[prime] splice acceptor site along with its 5[prime] UTR. The ability of the 3[prime] UTR of the [beta]-tubulin gene to maintain mRNA stability upon heat shock was compared to that of the 3[prime] UTR of the hsp 70 genes. Four different hph fusion genes were constructed (Fig.
Figure 3. Physical maps of different constructs and the hph locus in different transformed cell lines. (A) Structure of four plasmid constructs (P-H-T, H23-H-T, P-H-H2 and H23-H-H2). PARP, the PARP gene promoter and its 3[prime] splice acceptor site (24); H23, the hsp 70 intergenic region promoter and its 3[prime] splice acceptor site (21); Hph, hygromycin resistance gene; T, [beta][alpha]-tubulin intergenic region sequence (28); H2, the 3[prime] end of the hsp 70 gene 2; pUC, vector pUC18. The thick line in each construct represents the targeting sequence derived from the VSG 118 ES (see below). (B) Structure of the VSG 118 ES in different cell lines. Top: BC, structure of the basic copy VSG gene locus; ELC, structure of the VSG 118 ES (36). Bottom: structure of the hph-VSG 118 ES locus in four different cell lines (A, B, C and D). The EcoRI-HindIII fragment, underlined in ELC, was used as a targeting sequence. 118, VSG 118 gene; [phiv], the pseudo-VSG 118 gene (22); ESAG, expression site associated genes (36); double slashes, a distance of 10-20 kb; triple arrows, telomere repeats; flag, a functional promoter; flag with a cross, the inactivated VSG promoter of the VSG 118 ES; four closely arranged vertical lines, 70 bp repeats. The numbers below the parenthesis indicate the copy number of tandemly arranged input plasmid that were integrated into the target site. Restriction enzyme sites are indicated by E, EcoRI; H, HindIII; P, PvuII; S, SalI. Hybridization probes used in the Southern blot analysis and PFGE are indicated above the physical map: HP, the HindIII-PvuII fragment spanning the 3[prime] end of the pseudo-VSG 118; VSG, the 5[prime] coding region of the VSG 118 gene. The dashed lines indicated the PvuII fragments detected by probe HP. Others as described in (A). (C) Structure of the hph-hsp 70 locus in cell line E. Boxes indicated with 1-6 represent the hsp 70 genes 1-6. The hybridization probe Hsp indicated above the physical map is derived from the HindIII fragment of the coding region of the hsp 70 gene 3. The dashed lines indicated the PvuII fragments detected by probe Hsp. Others are described in (A) and (B). Construct P-H-T encodes the hph gene (H) which is controlled by the PARP promoter (P) and flanked by a PARP 3[prime] splice acceptor site, and the [beta]-[alpha] tubulin intergenic region (T), providing for trans-splicing and polyadenylation signals, respectively. Construct H23-H-T encodes the hph gene under the control of an hsp 70 intergenic region promoter and a 3[prime] splice acceptor site from the 5[prime] UTR of the hsp 70 gene (H23; construct H23-H-T is identical to the previously described construct H23H-B7; 21). At its 3[prime] end, the hph gene is flanked by the [beta]-[alpha] tubulin intergenic region. In construct H23-H-H2, the hph gene is flanked at its 5[prime] and 3[prime] end, respectively, by the hsp 70 intergenic region promoter, the hsp 70 3[prime] splice acceptor site and the 3[prime] end of hsp 70 gene 2 (H2) which provides a polyadenylation signal. In construct P-H-H2, the hph gene is flanked at its 5[prime] end, by the PARP promoter, PARP 3[prime] splice acceptor site (P), and at its 3[prime] end, by the 3[prime] end of hsp 70 gene 2. A DNA fragment encoding sequences derived from the VSG 118 expression site (ES) located downstream of the large 70 bp array and upstream of a VSG pseudo gene was added downstream of the hph transcription unit in each construct, to direct targeting of plasmid DNA into the VSG 118 ES in 118 clone 1 procyclic trypanosome (Fig. These four constructs were linearized at a SalI restriction enzyme site in the center of VSG 118 ES sequences and electroporated into the 118 clone 1 procyclic trypanosomes. Following selection with hygromycin B, individually transformed trypanosomes were isolated. Physical mapping of the hph locus in each cell line confirmed the predicted integration events (Figs The integration of plasmid DNA into the VSG 118 ES and the hsp 70 locus in cell lines A, C, D and E, respectively, was further confirmed by PFGE analysis (Fig. 
Figure 4. Structural analysis of the hph locus in transformed trypanosome cell lines. (A) Southern genomic blot analysis. Genomic DNAs from wild type trypanosomes (W) and five different transformed cell lines (A, B, C, D and E) were digested with the restriction enzyme PvuII, separated in 0.8% agarose gels and transferred onto nitrocellulose filters. The final post-hybridizational washes were performed in 0.1× SSC, 0.1% SDS at 65°C. Arrowheads indicate the VSG ES associated fragments. Dots indicate the polymorphic DNA fragment generated by the integration of input plasmid into the hsp 70 locus in cell line E. The hybridization probe used is indicated on the top of each panel. HP and Hsp, as indicated in Figure 3; Hph, the hph coding region. (B) Chromosomal location of the hph gene in transformed trypanosomes. PFGE analysis and chromosome assignments in T.brucei were performed as previously described (26,27). Chromosome blocks of wild type trypanosomes (W) and different cell lines (A, C, D and E) were separated in a 1% agarose gel for 7 days at 3 V/cm and a pulse frequency of 3300 s. Parallel PFGE Southern blots were hybridized with the Hph probe and the VSG 118 5[prime] coding region (VSG) probe. The final post-hybridizational washes were performed in 0.1× SSC, 0.1% SDS at 65°C. 10, 14 and 19[prime] indicate chromosome bands 10, 14 and 19[prime] respectively, as described (26,27).
A
B
Temperature effects on the steady mRNA levels in different transformed cell lines
All transformed trypanosome cell lines were normally maintained at ~25°C. Aliquots of cultured trypanosomes from each cell line were incubated at 41°C for 30, 60 or 180 min. Total RNAs were isolated from heat shocked trypanosomes and trypanosomes maintained at standard conditions. Then the level of steady state mRNAs encoded by different genes in response to the 41°C heat shock was determined by northern blot analysis (Fig.
I further addressed whether the 3[prime] UTR can affect mRNA levels upon heat shock. In cell line C, which was generated with the P-H-H2 construct, the level of hph mRNA, surprisingly, stayed constant upon heat shock as it occurred with that of the endogenous hsp 70 mRNA, even though the transcription of the hph gene was driven by the PARP promoter whose efficiency was almost abolished under heat shock (14). In contrast, the steady state mRNA of [beta]-tubulin was again drastically decreased upon heat shock at 41°C (Fig.
Figure 5. Comparison of steady state mRNAs derived from different cell lines in response to elevated temperatures. RNA samples were isolated from five different transformed trypanosome cell lines (A, B, C, D and E) that were maintained at 25°C (1) and incubated at 41°C for 30 min (2), 60 min (3) and 180 min (4), respectively. RNA samples were separated on 1% formaldehyde-agarose gels and transferred to nitrocellulose filters. The filter was sequentially hybridized with different 32P-labeled probes as indicated on the top of each panel. Signals on the filter were removed prior to hybridization with a different probe. Following hybridizations, the filters were washed to a final stringency of 65°C, 0.1× SSC and 0.1% SDS. Probes used are indicated by Hsp, the 0.8 kb HindIII fragment derived from the hsp 70 gene coding region (20); Tub, Hph and rRNA probes are as described in Figures 2 and 4. 2.4 and 1.3 indicate the position of 2400 and 1300 nt size standards. To determine whether the 3[prime] processing (i.e. polyadenylation) of the hph mRNAs was carried using the predicted polyadenylation signals residing in either the [beta][alpha] tubulin intergenic region or the hsp 70 gene intergenic region, the 3[prime] ends of hph mRNAs were determined by nucleotide sequence analysis of 3[prime] end cDNAs generated by RT-PCR. Nucleotide sequences of three individually picked clones from each batch of 3[prime] end cDNAs were analyzed. The results indicated that the hph mRNAs bearing the tubulin 3[prime] UTR are polyadenylated in the region where polyadenylation of endogenous [beta]-tubulin mRNAs occurred; these polyadenylation sites are located at 368-388 bp downstream of the TAA codon of the [beta]-tubulin gene, as described by Matthews et al. (42). In most clones analyzed, polyadenylation occurred after the TTT nucleotide sequence located at 386-388 bp downstream of the TAA codon of the [beta]-tubulin gene (referred to as site 1) indicated in Figure Figure 6. Mapping the polyadenylation sites of the hph mRNAs generated from different cell lines. The 3[prime] end cDNAs encoding the hph mRNAs were obtained by RT-PCR. The resulting cDNAs were subcloned into M13 vectors and their nucleotide sequences were analyzed. (A) The 3[prime] end sequences of the hph mRNA bearing the 3[prime] UTR of the [beta]-tubulin gene. The bold An and A indicate the polyadenylation sites used for the maturation of the hph mRNA. The italicized A residues indicate other alternative polyadenylation sites used for the maturation of endogenous tubulin mRNA (37). (B) The 3[prime] end sequence of the hph mRNA bearing the 3[prime] UTR of the hsp 70 gene. The bold An indicates the polyadenylated A residues. The results demonstrate that in T.brucei heat shock reduces steady state mRNA levels of many protein coding genes. An exception exists for the hsp 70 genes. By nuclear run-on analysis, it was previously shown that transcription of the hsp 70 and tubulin genes were about equally affected by a heat shock at 41°C (14). Thus, drastically different effects on steady state mRNA levels encoded by the hsp 70 and tubulin genes during heat shock are most likely not only due to a difference in the mRNA synthesis rates but mainly the result from changing the rate of RNA turnover. In higher eukaryotes, transcriptional efficiency of the hsp 70 genes is rapidly increased by heat shock, reaches a maximal level by 60 min and decreases thereafter. The continued synthesis of hsp 70 also requires the stability and efficient export of its mRNA at higher temperatures (9-13,39,40). Unlike the event in other eukaryotes, transcriptional induction of hsp 70 genes by heat shock does not occur in trypanosomes (14). The high level expression of the hsp 70 genes during heat shock is mainly post-transcriptionally controlled. Post-transcriptional control could be exerted at the level of RNA processing (trans-splicing and poly A addition), RNA transport and/or RNA stability. The presented results indicated that the 5[prime] end processing (trans-splicing) may be less important in the control of the mRNA level upon heat shock in T.brucei, while the importance of the 3[prime] end processing in the control of mRNA level during heat shock is not excluded. It is possible that in T.brucei, heat shock may also change the export efficiency of most mRNAs except heat shock mRNAs or a subset of mRNAs, as the events described in yeast (40). We are currently investigating these possibilities. A drastically increased expression of hsp 70 mRNA upon heat shock can be detected in some stocks of the procyclic trypanosome (for example the strain 427 RB). It is possible that the procyclic 427 RB strain may have altered its sensitivity to heat shock or other stress signals due to its long period of maintenance in the laboratory. It has been documented that pre-experienced environmental stress may affect the level of heat shock response (15). Thus, the extent to which heat shock can affect gene expression may vary in trypanosome strains of different origins. To exclude potential artefacts, I also examined the effect of temperature on the mRNA levels of trypanosomes cultured in different phases of their growth curve. Surprisingly, the steady state mRNA levels of tubulin and CRAM genes were less significantly reduced after heat shock at 41°C in trypanosomes of late log or stationary phase when compared to that in trypanosomes of early log and log phase. The level of the hsp 70 mRNA was, as expected, not significantly affected (not shown). It should be noted that, for some genes, the level of steady state mRNA was markedly reduced in trypanosomes of late log phase, as previously reported (15). Thus, to prevent potential artefacts, the early to mid log phase trypanosomes (cell density from 4 to 8 × 106 cells/ml) were used in all other experiments. It is currently being investigated whether the level of potential trans-acting factors that affect mRNA stability upon heat shock may be reduced in late log phase trypanosomes. Most genes in trypanosomes are tightly clustered and polycistronically transcribed. As a result, the differential expression of steady state mRNA of individual genes derived from a single polycistronic precursor requires post-transcriptional control. Thus far, only expression of the VSG and PARP genes appears to involve both transcriptional and post-transcriptional controls, while other differentially expressed protein coding genes analyzed in trypanosomes rely on post-transcriptional controls (41,42). Post-transcriptional control of mRNA abundance seems to play a major role in the regulation of many differentially expressed genes in T.brucei (for a review see 42). The mechanism of post-transcriptional control in trypanosomes is not yet completely clear. However, it has been shown that trans-splicing and poly A addition may be coupled and the selection of the poly A addition site is specified by the location of the downstream 3[prime] splice acceptor site (37,38). Apparently, removal of the characteristic polypyrimidine tract of the intergenic region significantly reduced the efficiency of both the trans-splicing and polyadenylation, resulting in a low level of mRNA (37). In T.brucei, the maturation of hsp 70 mRNA can occur by using three different splice acceptor sites (4). It was postulated that preferential usage of a particular splice acceptor site may correlate with the abundance of mRNA synthesized during heat shock. I can now exclude this model, since the hsp 70 promoter with its 3[prime] splice acceptor site and 5[prime] UTR was not able to up-regulate or maintain the mRNA level upon heat shock. It seems that mRNA stability plays a major role in the control of differential gene expression in the life cycle of trypanosomes. It was consistently found that nucleotide sequences responsible for the post-transcriptional regulation of mRNA abundance reside in the 3[prime] UTR of the gene (43-45). Similar control mechanisms for gene expression were also found in related parasites such as the hsp 83 gene of Leishmania (46-48) and the Amastin/Tuzin gene cluster of Trypanosoma cruzi (49). It has been reported that the 3[prime] UTR from various T.cruzi stage specific and constitutively expressed genes mediates the level of the steady state mRNA and/or the level of translation efficiency of the mRNA (50). I show that the 3[prime] end of the hsp 70 gene is the only sequence responsible for the maintenance of a high level of the steady state mRNA during heat shock. The hsp 70 intergenic region promoter, its 3[prime] splice acceptor sites and the 5[prime] UTR do not seem to affect the abundance of the mRNA. In contrast, in Leishmania, both the 5[prime] and 3[prime] UTR of the hsp 83 gene are required for control of the mRNA level at altered temperatures (46). Thus different sequences requirements may be involved in the post-transcriptional control of the hsp gene expression in different protozoan parasites. mRNA degradation plays an important role in the regulation of gene expression in eukaryotes (51,52). This process can be significantly influenced by exogenous factors. Many studies have shown that one of the mRNA degradation pathways is dependent on deadenylation of mRNA. In some cases, deadenylation may lead to 3[prime]->5[prime] exonucleolytic activity. For some mRNAs, shortening of the 3[prime] poly A tail leads to decapping followed by 5[prime]->3[prime] exonucleolytic degradation of the transcript. Other degradation pathways are independent of deadenylation, and rely on sequence-specific endonucleolytic cleavage of the mRNA or on deadenylation independent decapping followed by 5[prime]->3[prime] exonucleolytic degradation. Since in T.brucei, the 3[prime] UTR of the hsp 70 gene is sufficient to maintain mRNA abundance during heat shock, it is possible that degradation of mRNA in T.brucei, during heat shock, may be initiated from the 3[prime] end of the molecule via a heat induced process. A similar deadenylation dependent degradation pathway as described in other eukaryotes may be operating in T.brucei. Specific cis-acting elements in the 3[prime] UTR of hsp 70 gene in conjunction with trans-acting factor may prevent initial degradation events at the 3[prime] end and thus prevent other subsequent degradation processes. Comparing sequences immediately adjacent to the polyadenylation site of many different genes, a unique sequence of TTA repeats was found immediately upstream of the poly A addition site in the hsp 70 genes of T.brucei. This TTA repeat is also present in the intergenic region of the T.cruzi hsp 60, 70 and 83 genes (53-56). In the constitutively expressed hsp 70 gene-5 of Leishmania major, the poly A addition site is located immediately downstream of a TA repeat (57). We are currently investigating mechanisms of mRNA decay upon heat shock in T.brucei and the possible role of the UA-rich repeats of the hsp 70 3[prime] UTR in the maintenance of mRNA abundance. I thank Drs M.Eiki, M.Muranjan and L.H.T.Van der Ploeg for critical reading of the manuscript. This work was supported by NIH grant AI28953 to G.-S.M.L. who is a Burroughs Wellcome Fund New Investigator in Molecular Parasitology.
Determination of the 3[prime] ends of hph mRNAs
DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES
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