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• W2000529632 abstract "We recently characterized a large developmentally regulated gene family in Leishmania encoding the amastin surface proteins. While studying the regulation of these genes, we identified a region of 770 nucleotides (nt) within the 2055-nt 3′-untranslated region (3′-UTR) that regulates stage-specific gene expression at the level of translation. An intriguing feature of this 3′-UTR regulatory region is the presence of a ∼450-nt element that is highly conserved among several Leishmania mRNAs. Here we show, using a luciferase reporter system and polysome profiling experiments, that the 450-nt element stimulates translation initiation of the amastin mRNA in response to heat shock, which is the main environmental change that the parasite encounters upon its entry into the mammalian host. Deletional analyses depicted a second region of ∼100 nucleotides located at the 3′-end of several amastin transcripts, which also activates translation in response to elevated temperature. Both 3′-UTR regulatory elements act in an additive manner to stimulate amastin mRNA translation. In addition, we show that acidic pH encountered in the phagolysosomes of macrophages, the location of parasitic differentiation, triggers the accumulation of amastin transcripts by a distinct mechanism that is independent of the 450-nt and 100-nt elements. Overall, these important findings support the notion that stage-specific post-transcriptional regulation of the amastin mRNAs in Leishmania is complex and involves the coordination of distinct mechanisms controlling mRNA stability and translation that are independently triggered by key environmental signals inducing differentiation of the parasite within macrophages. We recently characterized a large developmentally regulated gene family in Leishmania encoding the amastin surface proteins. While studying the regulation of these genes, we identified a region of 770 nucleotides (nt) within the 2055-nt 3′-untranslated region (3′-UTR) that regulates stage-specific gene expression at the level of translation. An intriguing feature of this 3′-UTR regulatory region is the presence of a ∼450-nt element that is highly conserved among several Leishmania mRNAs. Here we show, using a luciferase reporter system and polysome profiling experiments, that the 450-nt element stimulates translation initiation of the amastin mRNA in response to heat shock, which is the main environmental change that the parasite encounters upon its entry into the mammalian host. Deletional analyses depicted a second region of ∼100 nucleotides located at the 3′-end of several amastin transcripts, which also activates translation in response to elevated temperature. Both 3′-UTR regulatory elements act in an additive manner to stimulate amastin mRNA translation. In addition, we show that acidic pH encountered in the phagolysosomes of macrophages, the location of parasitic differentiation, triggers the accumulation of amastin transcripts by a distinct mechanism that is independent of the 450-nt and 100-nt elements. Overall, these important findings support the notion that stage-specific post-transcriptional regulation of the amastin mRNAs in Leishmania is complex and involves the coordination of distinct mechanisms controlling mRNA stability and translation that are independently triggered by key environmental signals inducing differentiation of the parasite within macrophages. The protozoan parasite Leishmania constitutes a major health problem in several endemic tropical and sub-tropical regions around the world, threatening over 350 million people of which more than 15 million are infected (1Ashford R.W. Desjeux P. de Raadt P. Parasitol. Today. 1996; 8: 104-105Abstract Full Text PDF Scopus (337) Google Scholar, 2Ashford R.W. Int. J. Parasitol. 2000; 30: 1269-1281Crossref PubMed Scopus (421) Google Scholar). At least 20 different Leishmania species are responsible for the various clinical manifestations of leishmaniasis, ranging from chronic skin ulcers (L. major, L. tropica, and L. mexicana) to more severe naso-pharynx mucosal destruction (L. braziliensis) or life-threatening visceral diseases (L. donovani, L. infantum, and L. chagasi) (3Herwaldt B.L. Lancet. 1999; 354: 1191-1199Abstract Full Text Full Text PDF PubMed Scopus (1359) Google Scholar). No effective vaccine is currently available against Leishmania infections, and resistance to the main anti-leishmanial agents is dramatically increasing in several endemic areas (4Sundar S. Trop. Med. Int. Health. 2001; 6: 849-854Crossref PubMed Scopus (485) Google Scholar). These facts have underscored the urgency of identification of new drug targets for chemotherapy and/or vaccine development.The life cycle of Leishmania includes two developmental stages: the extracellular promastigote form, transmitted to the mammalian host by the sand fly vector, and the amastigote form, adapted to resist and replicate within the threatening environment of the phagolysosomes. This adaptation requires a dynamic process implicating morphological and physiological changes within the parasite (5Glaser B. Gothe R. Tierarztl. Prax. Ausg. K. Klientiere. Heimtiere. 1998; 26: 40-46PubMed Google Scholar, 6Goyard S. Segawa H. Gordon J. Showalter M. Duncan R. Turco S.J. Beverley S.M. Mol. Biochem. Parasitol. 2003; 130: 31-42Crossref PubMed Scopus (144) Google Scholar, 7MacFarlane 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, 8Turco S.J. Sacks D.L. Mol. Biochem. Parasitol. 1991; 45: 91-99Crossref PubMed Scopus (77) Google Scholar, 9Zilberstein D. Shapira M. Annu. Rev. Microbiol. 1994; 48: 449-470Crossref PubMed Scopus (315) Google Scholar) that are mainly orchestrated by the differential expression of a variety of genes. To date, several amastigote-specific genes have been characterized in Leishmania (reviewed in Ref. 10Papadopoulou B. Boucher N. McNicoll F. Wu Y. Dubé M. El-Fakhry Y. Huang X.F. J. Parasitol. 2003; 89: S174-S181Crossref PubMed Scopus (9) Google Scholar). Due to the absence of transcriptional control in Leishmania, stage-specific regulation of gene expression occurs exclusively at the post-transcriptional level (reviewed in Ref. 11Clayton C.E. EMBO J. 2002; 21: 1881-1888Crossref PubMed Scopus (447) Google Scholar) and involves mainly sequences within the 3′-UTR 5The abbreviations used are: UTRuntranslated regionntnucleotide(s). 5The abbreviations used are: UTRuntranslated regionntnucleotide(s). of mRNAs that determine mRNA abundance by modulating RNA stability (12Aly R. Argaman M. Halman S. Shapira M. Nucleic Acids Res. 1994; 22: 2922-2929Crossref PubMed Scopus (72) 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, 14Wu Y. El-Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (87) Google Scholar) or translational efficiency (15Boucher N. Wu Y. Dumas C. Dube M. Sereno D. Breton M. Papadopoulou B. J. Biol. Chem. 2002; 277: 19511-19520Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 16Larreta R. Soto M. Quijada L. Folgueira C. Abanades D.R. Alonso C. Requena J.M. BMC Mol. Biol. 2004; 5: 3Crossref PubMed Scopus (52) Google Scholar, 17Zilka A. Garlapati S. Dahan E. Yaolsky V. Shapira M. J. Biol. Chem. 2001; 276: 47922-47929Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). However, the molecular mechanisms underlying stage-specific regulation of gene expression in this parasite are not yet understood. Recent microarray analyses suggested that only ∼1–2% of L. major genes show significant changes in their mRNA levels (2-fold or more) throughout the life cycle of the parasite (18Akopyants N.S. Matlib R.S. Bukanova E.N. Smeds M.R. Brownstein B.H. Stormo G.D. Beverley S.M. Mol. Biochem. Parasitol. 2004; 136: 71-86Crossref PubMed Scopus (96) Google Scholar, 19Saxena A. Worthey E.A. Yan S. Leland A. Stuart K.D. Myler P.J. Mol. Biochem. Parasitol. 2003; 129: 103-114Crossref PubMed Scopus (80) Google Scholar). Similar data were obtained with the related parasite Trypanosoma brucei, where only ∼2% of mRNAs showed more than 2-fold differences in expression (20Diehl S. Diehl F. El-Sayed N.M. Clayton C. Hoheisel J.D. Mol. Biochem. Parasitol. 2002; 123: 115-123Crossref PubMed Scopus (57) Google Scholar). However, recent proteomic analyses revealed that ∼9% of the Leishmania proteins are differentially expressed in the amastigote stage (21Bente M. Harder S. Wiesgigl M. Heukeshoven J. Gelhaus C. Krause E. Clos J. Bruchhaus I. Proteomics. 2003; 3: 1811-1829Crossref PubMed Scopus (128) Google Scholar, 22El-Fakhry Y. Ouellette M. Papadopoulou B. Proteomics. 2002; 2: 1007-1017Crossref PubMed Scopus (94) Google Scholar). 6F. McNicoll and B. Papadopoulou, in preparation. 6F. McNicoll and B. Papadopoulou, in preparation. This difference between large-scale transcriptomic and proteomic studies further outlines the importance of translational and post-translational control in regulating gene expression in this organism.We previously identified a developmentally regulated gene family in Leishmania encoding the amastin surface proteins (14Wu Y. El-Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (87) Google Scholar) that share homology with the T. cruzi amastin proteins (23Teixeira S.M. Russell D.G. Kirchhoff L.V. Donelson J.E. J. Biol. Chem. 1994; 269: 20509-20516Abstract Full Text PDF PubMed Google Scholar). We recently characterized this large gene family in Leishmania and showed that it comprises up to 45 members, the majority of which are specifically expressed in the intracellular amastigote stage of the parasite (24Rochette A. McNicoll F. Girard J. Breton M. Leblanc E. Bergeron M.G. Papadopoulou B. Mol. Biochem. Parasitol. 2005; 140: 205-220Crossref PubMed Scopus (79) Google Scholar). Developmental regulation of the amastin transcripts in Leishmania (14Wu Y. El-Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (87) Google Scholar, 15Boucher N. Wu Y. Dumas C. Dube M. Sereno D. Breton M. Papadopoulou B. J. Biol. Chem. 2002; 277: 19511-19520Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) as well as in T. cruzi (25Teixeira S.M. Kirchhoff L.V. Donelson J.E. J. Biol. Chem. 1995; 270: 22586-22594Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) is mediated by defined regions within the 3′-UTR that are different in size and in sequence composition. While studying one of the L. infantum amastin gene homologs, we delimited a region of 770 nt within the 3′-UTR that regulates stage-specific gene expression most likely at the level of translation (15Boucher N. Wu Y. Dumas C. Dube M. Sereno D. Breton M. Papadopoulou B. J. Biol. Chem. 2002; 277: 19511-19520Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). An intriguing feature of this 3′-UTR regulatory region is the presence of a ∼450-nt element that is highly conserved not only among the majority of the amastin transcripts (24Rochette A. McNicoll F. Girard J. Breton M. Leblanc E. Bergeron M.G. Papadopoulou B. Mol. Biochem. Parasitol. 2005; 140: 205-220Crossref PubMed Scopus (79) Google Scholar) but also among several other Leishmania mRNAs, including known amastigote-specific mRNAs (15Boucher N. Wu Y. Dumas C. Dube M. Sereno D. Breton M. Papadopoulou B. J. Biol. Chem. 2002; 277: 19511-19520Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Here, we show that the 450-nt element stimulates translation of the amastin mRNAs in response to heat shock, which is the major environmental change that the parasite encounters upon its transmission from the sandfly to the mammalian host. Furthermore, we show that a second region of ∼100 nt located at the 3′-end of several amastin transcripts activates also amastin translation in response to elevated temperature. Importantly, we describe here that acidic pH encountered in the phagolysosomes, the location of differentiation of the parasite, has no effect on amastin translational regulation, but it triggers the accumulation of amastin transcripts specifically in the amastigote stage by a distinct mechanism that is independent of the 450-nt and 100-nt-mediated translational control. Overall, our findings support the notion that stage-specific post-transcriptional regulation of the amastin transcripts in Leishmania is complex and involves the coordination of different mechanisms that are independently triggered by key environmental signals inducing promastigote to amastigote differentiation within macrophages.EXPERIMENTAL PROCEDURESCell Lines, Leishmania Culture, and Macrophage Infections in Vitro— The Leishmania infantum MHOM/MA/67/ITMAP-263 strain used in this study was described previously (26Sereno D. Cavaleyra M. Zemzoumi K. Maquaire S. Ouaissi A. Lemesre J.L. Antimicrob. Agents Chemother. 1998; 42: 3097-3102Crossref PubMed Google Scholar). Promastigotes were cultured at pH 7.0 and 25 °C in SDM-79 medium supplemented with 10% heat-inactivated fetal calf serum (Multicell, Wisent Inc.) and 5 μg/ml hemin. L. infantum promastigote to amastigote differentiation in a host-free culture and the maintenance of axenic amastigotes were performed as previously described (22El-Fakhry Y. Ouellette M. Papadopoulou B. Proteomics. 2002; 2: 1007-1017Crossref PubMed Scopus (94) Google Scholar, 27Sereno D. Lemesre J.L. Antimicrob. Agents Chemother. 1997; 41: 972-976Crossref PubMed Google Scholar). Briefly, late stationary phase promastigotes (in average 6-day promastigotes) were inoculated in MAA/20 medium in 25-cm2-ventilated flasks and grown at 37 °C and pH 5.8 with 5% CO2 for ∼5 days until they fully differentiated to amastigote-like forms. Axenic amastigotes remained stable in culture, and after two to three passages they were recycled back to promastigotes to maintain their pathogenic potential. Axenic amastigotes grown under these conditions show morphological, biochemical, and biological characteristics similar to those of in vivo isolated amastigotes and are capable of infecting macrophages (28El Fadili K. Messier N. Leprohon P. Roy G. Guimond C. Trudel N. Saravia N.G. Papadopoulou B. Legare D. Ouellette M. Antimicrob. Agents Chemother. 2005; 49: 1988-1993Crossref PubMed Scopus (107) Google Scholar). In vitro infections of the THP-1 human leukemia monocyte cell line with Leishmania stationary promastigotes were carried out as previously reported (14Wu Y. El-Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (87) Google Scholar, 29Sereno D. Roy G. Lemesre J.L. Papadopoulou B. Ouellette M. Antimicrob. Agents Chemother. 2001; 45: 1168-1173Crossref PubMed Scopus (92) Google Scholar). The luciferase activity of the recombinant parasites was determined in both stages of the parasite's life cycle essentially as described elsewhere (15Boucher N. Wu Y. Dumas C. Dube M. Sereno D. Breton M. Papadopoulou B. J. Biol. Chem. 2002; 277: 19511-19520Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 30Roy 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 (139) Google Scholar).DNA Constructs and Transfections—Expression vectors LUC and LUC-770 previously referred to as pSPYNEOαLUC-IR and pSPYNEOαLUC-770-IR were described elsewhere (14Wu Y. El-Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (87) Google Scholar, 15Boucher N. Wu Y. Dumas C. Dube M. Sereno D. Breton M. Papadopoulou B. J. Biol. Chem. 2002; 277: 19511-19520Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The remaining vectors expressing the LUC-chimeric transcripts listed in Fig. 1B were made as follows. Briefly, various parts of the 770-nt 3′-UTR regulatory region of the L. infantum LinJ34.0840 amastin gene homolog (14Wu Y. El-Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (87) Google Scholar, 15Boucher N. Wu Y. Dumas C. Dube M. Sereno D. Breton M. Papadopoulou B. J. Biol. Chem. 2002; 277: 19511-19520Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) were amplified by PCR using TaqDNA polymerase (Qiagen). PCR fragments were digested with BamHI and cloned into the BamHI site of vector pSPYNEOαLUC-IR downstream of the firefly luciferase (LUC) open reading frame. In plasmid LUC-550, a region of 550 nucleotides containing the conserved 450-nt element flanked by ∼40 and 60 nucleotides on either side was amplified using primers P5′-550 (5′-CGGGATCCCGCCTCGGGCCCCTCGC-3′) and P3′-550 (5′-CGGGATCCCGTGCCAGGAAAAACAG-3′). In vector LUC-450, the 450-nt element was amplified with P5′-450 (5′-GTGGATCCCTAACTACACTTTC-3′) and P3′-450 (5′-CTAGATCTGCGACGGACAAGTC-3′). In vector LUC-300, the last 300 nucleotides of the amastin 3′-UTR were amplified with primers P5′-300 (5′-ACTTGTCCGTCGCGGAGTAAG-3′) and P3′-300 (5′-CGGGATCCCGGAGGAACGGAGACAA-3′). In LUC-200R, the last 200 nucleotides of the amastin 3′-UTR were amplified using primers P5′-200R (5′-CGGGATCCCGTATACACTATACACATATGT-3′) and P3′-300. Primers P5′-300 and P3′-200L (5′-CGGGATCCCGCCGTCGCGGAGTAAG-3′) were used to amplify the first 200 nucleotides of the 300-nt region (LUC-200L). The vector 5′-UTR-LUC-770 was made by a three-step cloning. First, vector pGEM3-NEO (31Papadopoulou B. Roy G. Ouellette M. EMBO J. 1992; 11: 3601-3608Crossref PubMed Scopus (185) Google Scholar) was digested with SacI and SmaI and ligated to the SacI-NsiI 340-bp fragment containing the 5′-UTR sequence of the amastin mRNA (211 nt) (14Wu Y. El-Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (87) Google Scholar), amplified by PCR (5′-ATGTCCTCGCCACTCTCCCC-3′ and 5′-CTTCGGTAAATCCGCAACAG-3′), and to the LUC-770 PstI-PvuII fragment containing the LUC gene fused to the 770-nt region of the amastin 3′-UTR. The plasmid copy number in each transfectant was evaluated by Southern blot and PhosphorImager analysis, and it was found to be similar for the different LUC-containing constructs (data not shown). The BT1-LUC, BT1-LUC-770, and BT1-LUC-300 constructs (see Fig. 3A) were made as follows. Vectors LUC-770, LUC-300, and LUC control (see Fig. 1B) were digested with HpaI and HindIII, filled in with Klenow and subcloned into the unique NotI site (also filled in with Klenow) of vector pSP-BT1 that contains the open reading frame of the biopterin transporter 1 gene (BT1) (32Kündig C. Haimeur A. Légaré D. Papadopoulou B. Ouellette M. EMBO J. 1999; 18: 2342-2351Crossref PubMed Scopus (87) Google Scholar). For genomic integration into the BT1 locus, ∼2.5 μg of HpaI-HindIII digests (these enzymes cut on either side of BT1) containing the different targeting cassettes were transfected by electroporation into L. infantum as described elsewhere (31Papadopoulou B. Roy G. Ouellette M. EMBO J. 1992; 11: 3601-3608Crossref PubMed Scopus (185) Google Scholar). Approximately 10–20 μg of purified plasmid DNA (Qiagen) was used for transfections. Transfected cells were plated on SDM-79 (2X) medium with 1.5% agar and 0.01 mg/ml of G418 (Sigma), and individual clones were obtained after 2–3 weeks.FIGURE 3Genomic integration of the amastin 3′-UTR regulatory elements to evaluate their role in stage-specific post-transcriptional regulation. A, schematic representation of the targeting cassettes for the genomic integration of both 3′-UTR regulatory elements of the amastin mRNA (450 and 100 nt) (BT1-LUC-770) or of the 100-nt region alone (BT1-LUC-300) fused to the LUC coding region into the L. infantum biopterin transporter 1 (BT1) locus, which is constitutively expressed. BT1-LUC is the control construct lacking any 3′-UTR regulatory sequence. For genomic integration by homologous recombination, the above targeting cassettes were excised with HpaI and HindIII digestion and introduced as linear fragments into L. infantum by electroporation as described under “Experimental Procedures.” Positions of key restriction enzymes for analyzing the genotypes of the different transfectants are indicated. B, Southern blot analysis to verify correct integration of LUC-chimeric constructs into the BT1 locus. Genomic DNA from the different transfectants (clones were used here) was extracted, digested with PstI or NcoI, transferred onto nylon membrane, and hybridized to a probe specific for the BT1 gene. Homozygous BT1 mutants with the two BT1 alleles (the Leishmania genome is diploid) successfully disrupted by the LUC-chimeric constructs were obtained in all three cases. C, Northern blot hybridization with a probe corresponding to the LUC coding region to evaluate the effect of both 3′-UTR regulatory elements (450 and 100 nt) on mRNA accumulation in both developmental stages of the parasite (P, promastigotes; A, amastigotes). The expression of the endogenous amastin transcript was verified by hybridization using a probe specific to the amastin 3′-UTR (14Wu Y. El-Fakhry Y. Sereno D. Tamar S. Papadopoulou B. Mol. Biochem. Parasitol. 2000; 110: 345-357Crossref PubMed Scopus (87) Google Scholar). Equal amounts of total RNA were used as indicated by ethidium bromide staining. D, luciferase reporter assays with L. infantum recombinant parasites expressing the integrated LUC-chimeric constructs grown as axenic amastigotes. The results are presented as the relative luciferase -fold increase compared with the control (BT1-LUC). Values are means ± S.E. of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nucleic Acid and Protein Manipulations—Genomic DNA was isolated with the DNAzol™ reagent. Total RNA of L. infantum promastigotes and axenic amastigotes was isolated using the TRIzol™ reagent (Invitrogen). Southern and Northern blot hybridizations were performed following standard procedures (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Leishmania cells were lysed and protein lysates were sonicated (10 pulses). The proteins were quantified using Amido Black 10B (Bio-Rad), and 50 μg of total protein lysates was loaded onto 10% SDS-PAGE gels for Western blot analyses. The gels were transferred on a polyvinylidene difluoride membrane (Immobilon-P, Millipore) and incubated for 1 h in blocking solution (phosphate-buffered saline with 0.1% Tween 20 and 5% nonfat dry milk). Then the first antibody (a goat anti-luciferase pAB, Promega) was added for 90 min in 1:1000 dilution. Following a few washes with phosphate-buffered saline supplemented with 0.1% Tween 20, a donkey anti-goat horseradish peroxidase conjugate antibody (Santa Cruz Biotechnology) diluted at 1:5000 was added for 60 min. After additional washes, the blot was visualized by chemiluminescence using a Renaissance kit (New Life Science Products). LUC RNA and protein levels were estimated by densitometric analyses using a PhosphorImager with ImageQuaNT 3.1 software. An anti-α-tubulin antibody (Sigma) was also used to verify equal protein loading on SDS-PAGE gels for Western blot analysis.Sucrose Gradient Analysis—Approximately 3 × 109 L. infantum axenic amastigotes grown up to late logarithmic phase were first incubated with 100 μg/ml cycloheximide (Sigma) for 10 min, washed with phosphate-buffered saline, and lysed with a Dounce homogenizer in lysis buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 10 mm MgCl2, 1 mm dithiothreitol, 0.5% IGEPAL, 100 μg/ml cycloheximide, 100 units/ml RNAguard (Amersham Biosciences), 1 mm phenylmethylsulfonyl fluoride, 15 μl/ml protease inhibitor mixture (Sigma)). Leishmania lysates were pelleted by centrifugation at 12,000 rpm for 15 min at 4 °C, and the supernatant (40 A260 nm units) was layered on top of a 15% to 45% linear sucrose gradient (10 ml) in gradient buffer (50 mm Tris-HCl, pH 7.4, 50 mm KCl, 10 mm MgCl2, 1 mm dithiothreitol, 3 units/ml RNAguard). The gradient was made with the Gradient Maker (#GM-100, CBS Scientific Co.). Ribosomal subunits (40 S and 60 S), monosomes (80 S), and polyribosomes were sedimented by centrifugation in a Beckman SW40 Ti rotor at 35,000 rpm for 2 h and 15 min at 4 °C as previously described (34Khandjian E.W. Corbin F. Woerly S. Rousseau F. Nat. Genet. 1996; 12: 91-93Crossref PubMed Scopus (206) Google Scholar, 35Mangus D.A. Jacobson A. Methods. 1999; 17: 28-37Crossref PubMed Scopus (53) Google Scholar). After centrifugation, approximately sixteen 0.6-ml fractions were collected at 4 °C using an ISCO Density Gradient Fractionation System under constant monitoring of absorption at 254 nm. The positions of the 40 S, 60 S, and 80 S and polysomal peaks were also corroborated by Northern blot analysis using an 18 S rRNA-specific probe. RNA was extracted from each fraction by phenol-chloroform and followed by ethanol precipitation and analyzed by Northern blot hybridization.RESULTSTwo Distinct 3′-UTR Elements Contribute to Stage-specific Regulation of the Amastin mRNA—We recently characterized a large family of developmentally regulated genes in Leishmania-encoding surface proteins, named amastins (24Rochette A. McNicoll F. Girard J. Breton M. Leblanc E. Bergeron M.G. Papadopoulou B. Mol. Biochem. Parasitol. 2005; 140: 205-220Crossref PubMed Scopus (79) Google Scholar). In one of the L. infantum amastin gene homologs that we have studied in more detail, we identified a region of 770 nt in the 3′-UTR (Fig. 1A) that regulates stage-specific gene expression most likely at the level of translation (15Boucher N. Wu Y. Dumas C. Dube M. Sereno D. Breton M. Papadopoulou B. J. Biol. Chem. 2002; 277: 19511-19520Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Remarkably, the first ∼450 nucleotides of this regulatory region (Fig. 1A) were found to be conserved among several known amastigote-specific mRNAs in Leishmania, including the majority of the amastin transcripts (15Boucher N. Wu Y. Dumas C. Dube M. Sereno D. Breton M. Papadopoulou B. J. Biol. Chem. 2002; 277: 19511-19520Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 24Rochette A. McNicoll F. Girard J. Breton M. Leblanc E. Bergeron M.G. Papadopoulou B. Mol. Biochem. Parasitol. 2005; 140: 205-220Crossref PubMed Scopus (79) Google Scholar). With the nucleotide sequence of the L. major and L. infantum genomes now completed, in silico screening depicted several hundreds of intergenic sequences that share variable levels of homology with the 450-nt element (data not shown). We initially hypothesized that this 450-nt conserved element was responsible for the 770-nt-mediated translational regulation of the amastin mRNA. To test this hypothesis, we made a series of deletions within the 770-nt region that we cloned downstream of the firefly luciferase (LUC) reporter gene (Fig. 1B). These LUC-chimeric constructs were transfected into Leishmania as part of episomal expression vectors, and LUC activities were measured in recombinant parasites grown in both developmental life stages using axenic culture systems and macrophage infections in vitro (Fig. 1B). As previously shown (15Boucher N. Wu Y. Dumas C. Dube M. Sereno D. Breton M. Papadopoulou B. J. Biol. Chem. 2002; 277: 19511-19520Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), the 770-nt region in construct LUC-770 increases LUC activity by more than 16-fold, specifically in amastigotes. The complete 450-nt element in vector LUC-450, surprisingly, confers low levels of LUC induction (∼2.5-fold). Recent BLAST analyses on the complete Leishmania genome (www.genedb.org/), and more careful sequence comparison studies between the 3′-UTRs of the 45 amastin gene homologs (24Rochette A. McNicoll F. Girard J. Breton M. Leblanc E. Bergeron M.G. Papadopoulou B. Mol. Biochem. Parasitol. 2005; 140: 205-220Crossref PubMed Scopus (79) Google Scholar) indicated that the region of homology extends by a few nucleotides of each side of the 450-nt element (data not shown). Based on this updated information, we made a new construct with 550 nt that contains ∼60 nt (5′-side) and ∼40 nt (3′-side) of either side of the 450-nt element. The LUC-550 construct confers higher induction of LUC activity (∼8-fold), which represents, however, half the regulation conferred by the 770-nt region (Fig. 1B). Extending the 3′-side by 50 more nucleotides did not increase further LUC activity (data not shown). These results suggest that the full-length element is required for regulation and that the extremities of the element may be critical for stabilizing an RNA secondary structure. Moreover, our deletion data suggest that other sequences within the 770-nt region located downstream of the 450-nt element may be involved in translational regulation of the amastin transcript.To examine this possibility, we fused the last ∼300 nt of the 770-nt region to the LUC coding region (LUC-300) and tested whether this sequence was capable of promoting the induction of LUC activity specifically in amastigotes. Indeed, the LUC-300 construct induces LUC activity by 8-fold, similarly to the levels of regulation by the 450-nt element in construct LUC-550 (Fig. 1B). A deletion of 100 nucleotides of either side of the 300-nt region in constructs LUC-200L (L for left side) and LUC-200R (R for right side), respectively, has no effect on regulation hence delimiting the second regulatory sequence of the amastin mRNA to an internal region of ∼100 nt (see Fig. 1B). As ind" @default.
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