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BLAST search (330-nt 3’UTR element)

BLAST search (330-nt 3’UTR element). - 3'UTR of ~47 L. major ORFs (65-96% identity). 3'UTR. 5'. 3'UTR. amastin homologue 24-3. 330nt. -Known amastigote-specific genes: ATPase 1b HSP100 Rab-14 GTP-binding protein Tcj1 protein Adenosine kinase Kinesin-like protein

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BLAST search (330-nt 3’UTR element)

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  1. BLAST search(330-nt 3’UTR element) -3'UTRof ~47 L. major ORFs (65-96% identity) 3'UTR 5' 3'UTR amastin homologue 24-3 330nt -Known amastigote-specific genes: ATPase 1b HSP100 Rab-14 GTP-binding protein Tcj1 protein Adenosine kinase Kinesin-like protein Kinesin-like protein k39 ADP/ATP carrier ANC ADP/ATP carrier Ser/thr protein kinase Mitogen activated protein kinase Ras-related protein rab-4 Phosphatase tRNA-Leu Met-tRNA-synthetase Met-aminopeptidase 2 Aminomethyltransferase S-Adenosyl-Methyl-transferase Geranyl-geranyldiphosphate synthase Zincfinger transmembrane protein DEAD/DEAH box helicase Transmembrane glycoprotein Hypothetical proteins Amastin gene homologues

  2. Correlation between the presence of the 3’UTR conserved elements and stage-specific regulation of gene expression P A P A P A P A P A P A P A P A P A P A -Known Leishmania amastigote-specific genes:A2, 5’ A2rel, HSP100, Histone H1, ATPase 1b, Rab 14 -Differentially expressed proteins identified by proteomic analysis

  3. 450-nt 150-nt 330-nt The conserved 3’UTR elements are involved in stage-specific regulation of gene expression Pro Ama (mo) LUC // LUC LmjF34-0500 LUC LUC LmjF34-1560 LUC // LUC LmjF24.1270 LUC // 1 2 3 4 5 6 7 8 9 10 18 Relative LUC fold increase

  4. Stage-specific regulation of the Ldi-3544 amastin mRNA IR amastin 3'UTR 5' amastin ORF 150-nt 450-nt

  5. P A B LUC-770 rRNA amastin The 770-nt 3’UTR regionof the amastin mRNA is not associated with mRNA abundance and stability

  6. P A B LUC-770 rRNA amastin The 770-nt 3’UTR regionof the amastin mRNA is not associated with mRNA abundance and stability The 770-nt 3’UTR region regulates mRNA translation Pro Ama LUC 61 kDa

  7. sucrose 15% 45% sucrose Ribosomal subunits 15% 45% Ribosomal subunits mRNP 80S 60S 40S Polysomes mRNP 80S 60S 40S Polysomes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Pro/LUC-770 Ama/LUC-770 LUC rRNA rRNA LUC rRNA The 770-nt of the amastin 3’UTR enhances mRNA translation in a stage-specific manner

  8. Pro Ama LUC-2 kb LUC-2 kb LUC-D450 LUC-D450 LUC-450 LUC-770 LUC-450 LUC-770 LUC-IR LUC-IR LUC a-tub Both conserved elements in the 3’UTR of the amastin mRNA participate in stage-specific translational control

  9. sucrose 15% 45% a-tub Both conserved elements in the 3’UTR of the amastin mRNA participate in stage-specific translational control Pro Ama LUC-2 kb LUC-2 kb LUC-D450 LUC-D450 LUC-450 LUC-770 LUC-450 LUC-770 LUC-IR LUC-IR LUC mRNP 80S 60S 40S Polysomes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 LUC-770 LUC-D450

  10. polysomes 80S 80S 60S 40S 60S polysomes 40S Promastigotes (26°C, pH 7.0) pH 5.5 + 37° C (1h) A combination ofheat and acidic pH stressdramatically decreases mRNA translation 80S 80S 60S polysomes 40S 60S polysomes 40S pH 5.5 + 37° C (O/N) pH 5.5 + 37° C (5h)

  11. Pulse labelling T° + pH 100000 80000 Incorporated 35S Met 60000 Control Stress 40000 20000 0 1 5 7 24 Time (hours) A combination ofheat and acidic pH stressdramatically decreases mRNA translation

  12. CONCLUSIONS STAGE-SPECIFIC TRANSLATIONAL CONTROL IN LEISHMANIA IS MEDIATED BY A VARIETY OF MECHANISMS -Distinct conserved elements are found in the 3'UTR of a large number of developmentally regulated mRNAs in Leishmania Common mechanism of stage-specific gene regulation -These 3'UTR elements are unlike any other regulatory elements identified thus far in eukaryotes-Novel mechanism(s)? -The 3'UTR elements share conserved structural motifs -These 3'UTR conserved elements regulate stage-specific gene expression by enhancing mRNA translation without affecting mRNA stability -Not all stage-specific mRNAs in Leishmania contain these 3'UTR elements High complexity:RESPONSE TO VARIOUS ENVIRONMENTAL STRESSES THROUGHOUT THE LIFE CYCLE OF THE PARASITE

  13. Acknowledgments Graduate students: François McNicoll Maxime Laverdière Annie Rochette Serge Cloutier Marie-Noëlle Chou Michaela Müller Postdoctoral trainees: Dr. Conan Chow Research assistants: Carole Dumas Nathalie Boilard Canadian Institutes of Health Research Burroughs Wellcome Fund Collaborators: Dr. Marc Ouellette (U. Laval, CHUL) Dr. Jolyne Drummelsmith (U. Laval, CHUL) Dr. Antonis Koromilas (Lady Davis Institute) Denis Baltzis (Lady Davis Institute)

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