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A Common Mechanism of Stage-regulated Gene Expression in Leishmania Mediated by a Conserved 3′-Untranslated Region Element

2002; Elsevier BV; Volume: 277; Issue: 22 Linguagem: Inglês

10.1074/jbc.m200500200

1083-351X

Nathalie Boucher, Ying Wu, Carole Dumas, Marthe Dubé, Denis Séréno, Marie Breton, Barbara Papadopoulou,

Biochemical and Molecular Research

Developmental regulation of mRNA levels in trypanosomatid protozoa is determined post-transcriptionally and often involves sequences located in the 3′-untranslated regions (3′-UTR) of the mRNAs. We have previously identified a developmentally regulated gene family in Leishmania encoding the amastin surface proteins and showed that stage-specific accumulation of the amastin mRNA is mediated by sequences within the 3′-UTR. Here we identified a 450-nt region within the amastin 3′-UTR that can confer amastigote-specific gene expression by a novel mechanism that increases mRNA translation without an increase in mRNA stability. Remarkably, this 450-nt 3′-UTR element is highly conserved among a large number of Leishmania mRNAs in several Leishmania species. Here we show that several of these mRNAs are differentially expressed in the intracellular amastigote stage of the parasite and that the 450-nt conserved element in their 3′-UTRs is responsible for stage-specific gene regulation. We propose that the 450-nt conserved element, which is unlike any other regulatory element identified thus far, is part of a common mechanism of stage-regulated gene expression in Leishmania that regulates mRNA translation in response to intracellular stresses. Developmental regulation of mRNA levels in trypanosomatid protozoa is determined post-transcriptionally and often involves sequences located in the 3′-untranslated regions (3′-UTR) of the mRNAs. We have previously identified a developmentally regulated gene family in Leishmania encoding the amastin surface proteins and showed that stage-specific accumulation of the amastin mRNA is mediated by sequences within the 3′-UTR. Here we identified a 450-nt region within the amastin 3′-UTR that can confer amastigote-specific gene expression by a novel mechanism that increases mRNA translation without an increase in mRNA stability. Remarkably, this 450-nt 3′-UTR element is highly conserved among a large number of Leishmania mRNAs in several Leishmania species. Here we show that several of these mRNAs are differentially expressed in the intracellular amastigote stage of the parasite and that the 450-nt conserved element in their 3′-UTRs is responsible for stage-specific gene regulation. We propose that the 450-nt conserved element, which is unlike any other regulatory element identified thus far, is part of a common mechanism of stage-regulated gene expression in Leishmania that regulates mRNA translation in response to intracellular stresses. Parasites of the genus Leishmania cause cutaneous, mucocutaneous, and visceral infections affecting ∼400,000 people each year of the 397 million that are at risk worldwide (1Ashford R.W. Desjeux P. de Raadt P. Parasitol. Today. 1996; 8: 104-105Abstract Full Text PDF Scopus (337) Google Scholar). During its digenetic life cycle, Leishmania alternates between the alimentary tract of the sand fly vector as an extracellular promastigote and the acidic phagolysosomes of macrophages as an intracellular amastigote. Differentiation of the parasite into the amastigote form is a prerequisite for its intracellular survival. Several environmental factors including acidic pH, elevated temperature, and the harmful phagolysosomal milieu trigger cytodifferentiation accompanied by the differential expression of a variety of genes (2MacFarlane J. Blaxter M.L. Bishop R.P. Miles M.A. Kelly J.M. Eur. J. Biochem. 1990; 190: 377-384Crossref PubMed Scopus (105) Google Scholar, 3Glaser T.A. Moody S.F. Handman E. Bacic A. Spithill T.W. Mol. Biochem. Parasitol. 1991; 45: 337-344Crossref PubMed Scopus (58) Google Scholar, 4Turco S.J. Descoteaux A. Annu. Rev. Microbiol. 1992; 46: 65-94Crossref PubMed Google Scholar, 5Zilberstein D. Shapira M. Annu. Rev. Microbiol. 1994; 48: 449-470Crossref PubMed Scopus (313) Google Scholar, 6Garlapati S. Dahan E. Shapira M. Mol. Biochem. Parasitol. 1999; 100: 95-101Crossref PubMed Scopus (24) Google Scholar). Such stage-specific gene expression is crucial for adaptation because Leishmania differentiates from an extracellular to an intracellular parasite. Gene regulation in Leishmania and related trypanosomatids shares unique features that include polycistronic transcription of large RNA units by an α-amanitin-sensitive RNA polymerase II, probably in the absence of promoter elements, and pre-mRNA processing into monocistronic mRNAs through a post-transcriptional control mediated by trans-splicing and polyadenylation (7Borst P. Annu. Rev. Biochem. 1986; 55: 701-732Crossref PubMed Scopus (283) Google Scholar, 8Agabian N. Cell. 1990; 61: 1157-1160Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 9Myler P.J. Audleman L. deVos T. Hixson G. Kiser P. Lemley C. Magness C. Rickel E. Sisk E. Sunkin S. Swartzell S. Westlake T. Bastien P., Fu, G. Ivens A. Stuart K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2902-2906Crossref PubMed Scopus (208) Google Scholar).trans-Splicing and polyadenylation are mechanistically coupled in trypanosomatids and recognize regulatory signals that consist of polypyrimidine-rich sequences (10LeBowitz J.H. Smith H.Q. Rusche L. Beverley S.M. Genes Dev. 1993; 7: 996-1007Crossref PubMed Scopus (284) Google Scholar, 11Matthews K.R. Tschudi C. Ullu E. Genes Dev. 1994; 8: 491-501Crossref PubMed Scopus (216) Google Scholar). Numerous examples in Leishmania species support the notion that developmental regulation of mRNA levels is determined post-transcriptionally by sequences located in the 3′-untranslated regions (3′-UTR) 1The abbreviations used are: UTRuntranslated regionntnucleotide(s)LUCluciferaseCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidPBSphosphate-buffered salineIRintergenic region1The abbreviations used are: UTRuntranslated regionntnucleotide(s)LUCluciferaseCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidPBSphosphate-buffered salineIRintergenic region that usually control mRNA stability (12Aly R. Argaman M. Halman S. Shapira M. Nucleic Acids Res. 1994; 22: 2922-2929Crossref PubMed Scopus (71) Google Scholar, 13Charest H. Zhang W.W. Matlashewski G. J. Biol. Chem. 1996; 271: 17081-17090Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 14Beetham J.K. Myung K.S. McCoy J.J. Wilson M.E. Donelson J.E. J. Biol. Chem. 1997; 272: 17360-17366Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 15Burchmore R.J.S. Landfear S.M. J. Biol. Chem. 1998; 273: 29118-29126Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 16Quijada L. Soto M. Alonso C. Requena J.M. Mol. Biochem. Parasitol. 2000; 110: 79-91Crossref PubMed Scopus (45) Google Scholar, 17Wu Y., El- Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (85) Google Scholar). More recently, a novel mechanism of stage-specific regulation affecting pre-mRNA processing has been reported in Leishmania mexicana (18Brooks D.R. Denise H. Westrop G.D. Coombs G.H. Mottram J.C. J. Biol. Chem. 2001; 276: 47061-47069Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The role of 3′-UTRs and/or intercistronic regions in stage-specific gene regulation is further supported by the observation that differential expression of tandemly repeated genes is dependent on sequences present downstream of the different copies, which are often divergent within the same genomic locus (18Brooks D.R. Denise H. Westrop G.D. Coombs G.H. Mottram J.C. J. Biol. Chem. 2001; 276: 47061-47069Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 19Ramamoorthy R. Swihart K.G. McCoy J.J. Wilson M.E. Donelson J.E. J. Biol. Chem. 1995; 20: 12133-12139Abstract Full Text Full Text PDF Scopus (69) Google Scholar, 20Coulson R.M. Connor V. Chen J.C. Ajioka J.W. Mol. Biochem. Parasitol. 1996; 82: 227-236Crossref PubMed Scopus (48) Google Scholar, 21Quijada L. Soto M. Alonso C. Requena J.M. J. Biol. Chem. 1997; 272: 4493-4499Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Although several genes differentially expressed in the intracellular amastigote stage have been reported in Leishmania (14Beetham J.K. Myung K.S. McCoy J.J. Wilson M.E. Donelson J.E. J. Biol. Chem. 1997; 272: 17360-17366Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 17Wu Y., El- Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (85) Google Scholar, 21Quijada L. Soto M. Alonso C. Requena J.M. J. Biol. Chem. 1997; 272: 4493-4499Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 22Argaman M. Aly R. Shapira M. Mol. Biochem. Parasitol. 1994; 64: 95-110Crossref PubMed Scopus (83) Google Scholar, 23Charest H. Matlashewski G. Mol. Cell. Biol. 1994; 14: 2975-2984Crossref PubMed Scopus (137) Google Scholar, 24Mottram J.C. Frame M.J. Brooks D.R. Tetley L. Hutchison J.E. Souza A.E. Coombs G.H. J. Biol. 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Parasitol. 2000; 108: 93-99Crossref PubMed Scopus (28) Google Scholar), the molecular mechanisms that control developmental regulation in this organism are still not well understood. untranslated region nucleotide(s) luciferase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid phosphate-buffered saline intergenic region untranslated region nucleotide(s) luciferase 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid phosphate-buffered saline intergenic region We have recently identified a Leishmania gene family encoding the amastin surface proteins that are related to Trypanosoma cruzi amastins (29Teixeira S.M. Russell D.G. Kirchhoff L.V. Donelson J.E. J. Biol. Chem. 1994; 269: 20509-20516Abstract Full Text PDF PubMed Google Scholar) and showed that these genes are differentially expressed in the intracellular amastigote stage of the parasite and that the 3′-UTR of the amastin mRNA is required for increased mRNA accumulation in amastigotes (17Wu Y., El- Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (85) Google Scholar). We have now delineated a 450-nt region within the last third of the amastin 3′-UTR that confers stage-specific regulation and showed that this sequence is highly conserved among the 3′-UTRs of a large number of Leishmania mRNAs, several of which are known or shown in this study to be developmentally regulated in the mammalian-living form of the parasite. We show here that the 3′-UTR 450-nt conserved sequence can increase expression from a reporter mRNA in a stage-specific manner. Regulation by this 3′-UTR element does not increase mRNA abundance or stability, a major way of generating stage-specific gene expression in Leishmania and other trypanosomatids (12Aly R. Argaman M. Halman S. Shapira M. Nucleic Acids Res. 1994; 22: 2922-2929Crossref PubMed Scopus (71) Google Scholar, 13Charest H. Zhang W.W. Matlashewski G. J. Biol. Chem. 1996; 271: 17081-17090Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 14Beetham J.K. Myung K.S. McCoy J.J. Wilson M.E. Donelson J.E. J. Biol. Chem. 1997; 272: 17360-17366Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 15Burchmore R.J.S. Landfear S.M. J. Biol. Chem. 1998; 273: 29118-29126Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar,17Wu Y., El- Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (85) Google Scholar, 30Berberof M. Vanhamme L. Tebabi P. Pays A. Jefferies D. Welburn S. Pays E. EMBO J. 1995; 14: 2925-2934Crossref PubMed Scopus (97) Google Scholar, 31Furger A. Schurch N. Kurath U. Roditi I. Mol. Cell. Biol. 1997; 17: 4372-4380Crossref PubMed Scopus (135) Google Scholar, 32Hotz H.R. Hartmann C. Huober K. Hug M. Clayton C. Nucleic Acids Res. 1997; 25: 3017-3026Crossref PubMed Scopus (118) Google Scholar), but instead it increases protein levels, suggesting its implication in translational control. Our data point to a common mechanism of stage-specific regulation in Leishmania that might be utilized by a number of similarly regulated mRNAs. This is the first example of a common mechanism of stage-specific gene expression in protozoan parasites. Leishmania infantum LEM1317 and Leishmania major Friedlin MHOM/IL/80/FRIEDLIN strains have been described previously (33Lamontagne J. Papadopoulou B. J. Biol. Chem. 1999; 274: 6602-6609Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 34Tamar S. Dumas C. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 111: 401-414Crossref PubMed Scopus (4) Google Scholar).Leishmania promastigotes were cultured at pH 7.0 and 25 °C in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 5 mg/ml hemin. Adapted axenic amastigotes of L. infantum were maintained in MAA/20 medium at 37 °C in a 5% CO2 atmosphere as described previously (35Sereno D. Lemesre J.L. Antimicrob. Agents Chemother. 1997; 41: 972-976Crossref PubMed Google Scholar). L. major amastigotes were isolated from footpad lesions of infected BALB/c mice as described (36Muyombwe A. Olivier M. Harvie P. Bergeron M.G. Ouellette M. Papadopoulou B. J. Infect. Dis. 1998; 177: 188-195Crossref PubMed Scopus (32) Google Scholar). In vitroJ774 murine macrophage infections were done as indicated previously (17Wu Y., El- Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (85) Google Scholar, 37Sereno D. Roy G. Lemesre J.L. Papadopoulou B. Ouellette M. Antimicrob. Agents Chemother. 2001; 45: 1168-1173Crossref PubMed Scopus (91) Google Scholar). The luciferase (LUC) activity of the recombinant parasites was determined as reported previously (38Roy G. Dumas C. Sereno D., Wu, Y. Singh A.K. Tremblay M.J. Ouellette M., M., O. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 195-206Crossref PubMed Scopus (137) Google Scholar). The mean LUC activity was expressed in relative light units, and it was ranged from 400,000 to 700,000 in Leishmania grown as promastigotes, from 10,000 to 120,000 in axenic amastigotes, and from 500 to 12,000 in intramacrophage amastigotes. These marked differences in LUC activity depending on the medium and on the conditions of parasite growth are probably due to the extracellular acidic pH in which lysed amastigote cells are exposed and to the higher protease activity found in the phagolysosomes of the macrophages. It has been indeed reported that firefly luciferases are highly susceptible to acidic pH and to proteolysis (39Thompson J.F. Geoghegan K.F. Lloyd D.B. Lanzetti A.J. Magyar R.A. Anderson S.M. Branchini B.R. J. Biol. Chem. 1997; 272: 18766-18771Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 40Viviani V. Uchida A. Suenaga N. Ryufuku M. Ohmiya Y. Biochem. Biophys. Res. Commun. 2001; 280: 1286-1291Crossref PubMed Scopus (48) Google Scholar). The level of infection was also determined by optical microscopy examination following Diff Quick staining of cell preparation. Leishmania chromosomes were separated by clamped homogeneous electric field electrophoresis as described (41Dumas C. Ouellette M. Tovar J. Cunningham M.L. Fairlamb A.H. Tamar S. Olivier M. Papadopoulou B. EMBO J. 1997; 16: 2590-2598Crossref PubMed Scopus (271) Google Scholar). Total RNA of Leishmania cells was isolated using the guanidinium isothiocyanate method with TRIzol (Invitrogen). Southern and Northern blot hybridizations were performed following standard procedures (42Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Probes used in Fig. 5were amplified by PCR from L. major Friedlin genomic DNA using the following set of primers: P31_02-5′, 5′-ATGCAGCGCAGAATCAGCTCTA-3′, and P31_02-3′, 5′-AAACCCACTTGCGGGCACGA-3′ for the Lmflchr31_02 gene; P32_14-5′, 5′-GCTGTTGCGTTAGGTGGTGG-3′ and P32_14-3′: 5′-CCACCGCTGTGAAAACCAGA-3′ for the Lmflchr32_14 gene; and AL117263-P1, 5′-ACGCACCTGCAGGCGGTGTCCC-3′ and AL117263-P2, 5′-ACACTGCCCCTTCATCTGCC-3′ for the 3-ketoacyl-CoA thiolase gene. To prepare soluble protein lysates, Leishmania cells were harvested by centrifugation, washed with Hepes-NaCl, resuspended in lysis buffer (8 m urea, 4% CHAPS, 40 mmTris-base), and sonicated three times for 30 s. The proteins were quantified by the Amido Black 10B (Bio-Rad), and ∼35 μg of total protein extracts were loaded onto a 10% SDS-PAGE. The gels were transferred on a polyvinylidene difluoride membrane (Immobilon-P, Millipore) and blocked for 16 h with PBST (phosphate-buffered saline, 0.1% Tween 20, plus 1% gelatin solution). The first antibody, a goat anti-luciferase pAp (Promega) was diluted at 1:2000 in PBST with 1% gelatin and incubated 90 min with agitation. Following a few washes with PBST, a donkey anti-goat horseradish peroxidase conjugate antibody (Santa Cruz Biotechnology) diluted at 1:5000 in PBST with 1% gelatin was added and incubated for 60 min with the membrane. After additional washes, the final reaction was done with a Renaissance kit (New Life Science Products). LUC protein levels were estimated by densitometric analysis using a PhosphorImager with the ImageQuant 3.1 software. Expression vector pSPYNEOαLUC was made as described previously (17Wu Y., El- Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (85) Google Scholar). To construct vector pSPYNEOαLUC-IR, a 467-bp fragment (IR) containing the last 40 nucleotides of the amastin 3′-UTR with the natural poly(A) site (position 3250) and part of the intercistronic sequence between two amastin copies (see Fig. 1A) was amplified by PCR using Pwo DNA polymerase (Roche Molecular Biochemicals) and primers IR-5′ (5′-GCTTGCTTTTTGCTTTCTGTCA-3′) and IR-3′ (5′-GCGGCTCGCCAGTGTAGCAGA-3′) and subcloned into pSPYNEOαLUC digested with BamHI and filled in by Klenow fragment. To generate the different LUC-chimeric constructs listed in Fig. 1B, various parts of the 3′-UTR of the L. infantum amastin mRNA amplified by PCR by Pwo DNA polymerase and the following sets of primers (P35, 5′-GAGGAAATGAAGTGAAGGCG-3′ and P37, 5′-GAGGAACGGAGACAATAATG-3′ for amplifying the full-length 3′-UTR; P35 and P40, 5′-TTCCAGGCCTGCAGCGCACG-3′ for the first 415 nt; P35 and P41, 5′-CCTCGTCGTCCCCTCGATCA-3′ for the first 1000 nt; P37 and P44, 5′-GTGGCTGTCTAACTACACTT-3′ for the last 770 nt; P44 and P45, 5′-CCTTGTCTTTGCTCGTCCATTC-3′ for the 347-nt subregion within the 770 nt and primers P42, 5′-TGCGGCACGCACCTACACCA-3′ and P43, 5′-TAGCGGCCCGCCTTGTCTTTG-3′ for the 184-nt subregion) were subcloned into the BamHI site of pSPYNEOαLUC filled in by Klenow. The 467-bp fragment (IR) was introduced into Hin dIII site of the above LUC-chimeric vectors. To construct vectors pSPYNEOαLUC-A2 and pSPYNEOαLUC-A2–309 shown in Fig. 6, the full-length 3′-UTR of the Leishmania donovani A2 mRNA (A2-5′, 5′-GGCTCGGCGTCCGCTTTCCG-3′; A2-3′, 5′-TGCACTTTTCGTTTTTCCCGCA-3′) (23Charest H. Matlashewski G. Mol. Cell. Biol. 1994; 14: 2975-2984Crossref PubMed Scopus (137) Google Scholar) and the 309-nt region within the A2 3′-UTR (A2–309-5′, 5′-GCGGATCCCGGAAGCGTGGCGA-3′, A2–309-3′, 5′-CCGGATCCCACCACGAACAA-3′) that is homologous to the 450-nt region of the amastin 3′-UTR (see Fig. 4) were amplified using Pwo polymerase and the primers indicated above and subcloned downstream of the LUC gene into the BamHI site of vector pSPYNEOαLUC. Vector pSPYNEOαLUC-AC008242 (see Fig. 6) was generated by subcloning the PCR-amplified 394-bp region (Lm-ch27/5′, 5′-AAGCGCGACGAGAGCACCCT-3′, and Lm-ch27/3′, 5′-TCGAACAGGGCCATGCGTAT-3′) part of the SW3.1 L. major transcript showing homology to the 450-nt conserved element in the amastin 3′-UTR (see Fig. 4), into vector pSPYNEOαLUC digested with BamHI and filled in by Klenow. Finally, vector pSPYNEOαLUC-31_02 was constructed by subcloning a PCR-amplified fragment of 433 bp (31_02-5′, 5′-ACGCCAACGAGTTCTCCAGA-3′, and 31_02-3′, 5′-GCACAGCTCACCCCCGCCTC-3′) present in the 3′-UTR of the Lmflchr31_02 mRNA and shown 73% identity with the amastin 450-nt region into pSPYNEOαLUC as indicated above. The 467-bp IR sequence was then introduced downstream of these LUC chimeras as indicated above. 10–20 μg of purified plasmid DNA (Qiagen) was transfected into L. infantum by electroporation as described previously (43Papadopoulou B. Roy G. Ouellette M. EMBO J. 1992; 11: 3601-3608Crossref PubMed Scopus (185) Google Scholar). For the majority of the LUC chimeras corresponding to different parts of the amastin 3′-UTR fused to the LUC coding region, polyadenylation started at the amastin mRNA poly(A) site present in the 467-bp amplified IR sequence (see Fig. 1A), as determined by RT-PCR assays (data not shown).Figure 6The conserved 3′-UTR element in several amastigote-specific transcripts confers stage-specific gene regulation. The conserved region within the 3′-UTRs of A2, (309 nt), SW3.1 (394 nt), and the Lmflchr31_02 amastin homolog (433 nt) mRNAs, which is highly homologous to the amastin 450-nt element were subcloned downstream of the LUC reporter gene, transfected into L. infantum, and their role in amastigote-specific gene regulation was evaluated by measuring LUC activity in the transfectants grown under promastigote and axenic amastigote conditions. LUC assays were done in L. infantum, because it is not possible to axenically grow L. major amastigotes. The full-length A2 3′-UTR and the amastin intergenic sequence (IR; see Fig. 1A) were used as controls. 5′- and 3′-end processing of the LUC chimeric mRNAs is as described for Figs. 1 and 2. The results are presented as the relative luciferase fold increase compared with the control transfectant for each growth condition and are the averages of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Many amastigote-specific transcripts in Leishmania share a highly homologous 450-nt element in their 3′-UTRs.In silico screening using the 770-nt regulatory region of the amastin 3′-UTR as bait depicted a large number of Leishmania sequences displaying a 68–78% identity with the first 450 nucleotides of the 770-nt region. The homologous sequences within the 3′-UTRs of a selected number of amastigote-specific transcripts were aligned using the GCG Pileup program. These include the known L. donovani amastigote-specific genes HSP100 (26Krobitsch S. Brandau S. Hoyer C. Schmetz C. Hubel A. Clos J. J. Biol. Chem. 1998; 273: 6488-6494Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), A2 (13Charest H. Zhang W.W. Matlashewski G. J. Biol. Chem. 1996; 271: 17081-17090Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), and 5′A2rel (AC010851) (48Zhang W.W. Matlashewski G. Mol. Microbiol. 2001; 39: 935-948Crossref PubMed Scopus (94) Google Scholar), the L. major SW3.1 gene (AC008242) encoding histone H1 (25Noll T.M. Desponds C. Belli S.I. Glaser T.A. Fasel N.J. Mol. Biochem. Parasitol. 1997; 84: 215-227Crossref PubMed Scopus (38) Google Scholar), and the L. majorLmflchr31_02 and Lmflchr34_00 homologs of the amastin gene family and the 3-ketoacyl-CoA thiolase gene (AL117263) shown here to be differentially expressed in the intracellular amastigote stage of the parasite (see Fig. 5). The conserved region within the 3′-UTRs of the HSP100 and A2 amastigote-specific mRNAs was smaller than the average size of 450 nt found in a large number of Leishmania mRNAs (this figure and Table I). Nucleotide 1 corresponds to the position 2500 in the amastin mRNA, and nucleotide 454 corresponds to the position 2948 (see Fig. 1A and Ref. 17Wu Y., El- Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (85) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) RNA turnover was measured upon the addition of 10 μg/ml actinomycin D (Sigma) in both promastigote and axenic amastigote cultures of L. infantum-LUC recombinant transfectants as described previously (33Lamontagne J. Papadopoulou B. J. Biol. Chem. 1999; 274: 6602-6609Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Total RNA was extracted at various time points (0, 1, 3, and 5 h) following actinomycin D treatment. The RNA samples were subjected to Northern blot analysis using the LUC coding region as a probe. The levels of mRNA were normalized by hybridizing the blots with α-tubulin probe. We have recently identified a new developmentally regulated gene family in Leishmania, which shares a significant homology to the amastin surface proteins of T. cruzi and showed that stage-specific regulation of the amastin mRNA is mediated by sequences within the 3′-UTR (17Wu Y., El- Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (85) Google Scholar). To expand our studies on amastin gene developmental regulation, we made a series of chimeric constructs with regions spanning the 3′-UTR and cloned these sequences downstream of the luciferase reporter gene (LUC) (Fig. 1). To direct accurate 5′ and 3′ processing of the LUC chimeric transcripts, these cassettes were flanked by an upstream α-tubulin intergenic region and by a downstream region of 467 bp including the few last nucleotides of the amastin 3′-UTR with the natural poly(A) site of the amastin transcript followed by the amastin intergenic region (17Wu Y., El- Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (85) Google Scholar). In trypanosomatids, polyadenylation is often directed by trans-splicing signals that are located 100–400 nt downstream of the polyadenylation site (10LeBowitz J.H. Smith H.Q. Rusche L. Beverley S.M. Genes Dev. 1993; 7: 996-1007Crossref PubMed Scopus (284) Google Scholar, 11Matthews K.R. Tschudi C. Ullu E. Genes Dev. 1994; 8: 491-501Crossref PubMed Scopus (216) Google Scholar, 44Vassella E. Braun R. Roditi I. Nucleic Acids Res. 1994; 22: 1359-1364Crossref PubMed Scopus (80) Google Scholar, 45Schurch N. Hehl A. Vassella E. Braun R. Roditi I. Mol. Cell. Biol. 1994; 14: 3668-3675Crossref PubMed Scopus (86) Google Scholar). The LUC chimeras were subcloned into a neomycin phosphotransferase (NEO)-expression vector and introduced by electroporation into L. infantum cells (see "Experimental Procedures"). The LUC activity of the stable transfectants was evaluated in promastigotes, axenic amastigotes, and infected murine macrophages in vitro. We have previously shown (17Wu Y., El- Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (85) Google Scholar) and reconfirmed in this study that the full-length 3′-UTR of the amastin mRNA is able to increase LUC activity by 13–17-fold specifically in axenic and intramacrophage amastigotes, respectively (Fig. 1B). Deletion mutagenesis demonstrated that the first 1000 nucleotides of the amastin 3′-UTR are not associated with stage-specific regulation of LUC activity. However, the last 770 nucleotides of the amastin 3′-UTR are shown to induce LUC activity by 13-fold in axenic amastigotes and by 24.5-fold in intramacrophage amastigotes but not in promastigotes (Fig. 1B). Deletions spanning the 770-nt region of the amastin 3′-UTR significantly decreased regulation (Fig. 1B). Indeed, a subregion covering the first 347 nucleotides of the 770-nt region showed a 4–5-fold decrease in LUC activity in comparison with the LUC-770 construct (Fig. 1B). Further deletion within the 347-nt region giving rise to the LUC-184-nt chimeric construct has decreased LUC activity by another 50% (Fig. 1B). Thus, these deletion studies localized the cis-acting regulatory element responsible for stage-specific regulation of the amastin mRNA to a 350–770-nt region at the end of the amastin 3′-UTR. As previously reported for several amastigote-specific transcripts in Leishmania, mRNA abundance is mainly associated to a mechanism controlling mRNA turnover (13Charest H. Zhang W.W. Matlashewski G. J. Biol. Chem. 1996; 271: 17081-17090Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 17Wu Y., El- Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (85) Google Scholar, 22Argaman M. Aly R. Shapira M. Mol. Biochem. Parasitol. 1994; 64: 95-110Crossref PubMed Scopus (83) Google Scholar). Such an increase in mRNA stability was previously observed with the full-length 3′-UTR of the amastin mRNA (17Wu Y., El- Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (85) Google Scholar). We have now examined the role of the 770-nt regulatory region of the amastin 3′-UTR in inducing LUC mRNA stability in a stage-specific manner. To our surprise, the accumulation and/or the stability of the LUC -770 mRNA did not increase in the amastigote stage compared with promastigotes (Fig. 2), despite the fact that the 770-nt region is capable of inducing LUC activity by 25-fold specifically in amastigotes (Fig. 1B). Similarl