1. BLAST search (330-nt 3’UTR element) - 3'UTR of ~47 L. major ORFs (65-96% identity) 5' amastin homologue 24-3 3'UTR 330nt 3'UTR -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. The conserved 3’UTR elements are involved in stage-specific regulation of gene expression Relative LUC fold increase Pro Ama (mo) 1 2 3 4 5 6 7 8 9 10 18 450-nt 150-nt 330-nt LUC LmjF34-0500 LUC LmjF34-1560 LUC LmjF24.1270 //

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

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

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

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

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

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

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

11. Pulse labelling T° + pH 0 20000 40000 60000 80000 100000 15724 Time (hours) Incorporated 35 S Met Control Stress A combination of heat and acidic pH stress dramatically decreases mRNA translation

12. -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 STAGE-SPECIFIC TRANSLATIONAL CONTROL IN LEISHMANIA IS MEDIATED BY A VARIETY OF MECHANISMS CONCLUSIONS

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)