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 1bHSP100Rab-14

GTP-binding proteinTcj1 proteinAdenosine kinaseKinesin-like proteinKinesin-like protein k39ADP/ATP carrierANC ADP/ATP carrierSer/thr protein kinaseMitogen activated protein kinaseRas-related protein rab-4Phosphatase

tRNA-LeuMet-tRNA-synthetaseMet-aminopeptidase 2AminomethyltransferaseS-Adenosyl-Methyl-transferaseGeranyl-geranyldiphosphate synthaseZincfinger transmembrane proteinDEAD/DEAH box helicaseTransmembrane glycoproteinHypothetical proteinsAmastin gene homologues

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

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

LUC

LUC

LmjF34-1560

LUC

LUC

LUC

LmjF24.1270//

//

//

Stage-specific regulation of the Ldi-3544 amastin mRNA

amastin ORF amastin 3'UTR IR5'

450-nt 150-nt

The 770-nt 3’UTR regionof the amastin mRNA is not associated with

mRNA abundance and stabilityB

rRNA

LUC-770

amastin

P A

The 770-nt 3’UTR regionof the amastin mRNA is not associated with

mRNA abundance and stabilityB

rRNA

LUC-770

amastin

P A

Pro Ama

LUC61 kDa

The 770-nt 3’UTR region regulates mRNA translation

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

rRNArRNA

45%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

60S40S 80S

45% Ribosomal

subunits

15%sucrose

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Polysomes

mRNP

60S40S 80S

Ribosomal

subunits

15%sucrose

Polysomes

mRNP

Both conserved elements in the 3’UTR of the amastin mRNA participate in stage-specific translational control

LU

C-2

kb

Pro Ama

LU

C-2

kb

LU

C-7

70

LU

C-I

R

LU

C-

450

LU

C-4

50

LUC

LU

C-7

70

LU

C-I

R

LU

C-

450

LU

C-4

50tub

Both conserved elements in the 3’UTR of the amastin mRNA participate in stage-specific translational control

LUC-770

LUC-450

60S40S 80S

45%15%sucrose

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

PolysomesmRNP

LU

C-2

kb

LU

C-2

kb

LU

C-7

70

LU

C-I

R

LU

C-

450

LU

C-4

50

LUC

LU

C-7

70

LU

C-I

R

LU

C-

450

LU

C-4

50tub

Pro Ama

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

40S60S

80S

polysomes

pH 5.5 + 37° C (5h)

40S

60S80S

polysomes

40S

60S

80S

polysomes

Pulse labelling T° + pH

0

20000

40000

60000

80000

100000

1 5 7 24

Time (hours)

Inco

rpo

rate

d

35S

Met

Control

Stress

A combination of heat and acidic pH stress dramatically decreases mRNA translation

-Distinct conserved elements are found in the 3'UTR of a large number of developmentally regulated mRNAs in LeishmaniaCommon 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

Acknowledgments

Graduate students:

François McNicoll Maxime Laverdière

Annie RochetteSerge CloutierMarie-Noëlle ChouMichaela Müller

Postdoctoral trainees:

Dr. Conan Chow

Research assistants:

Carole Dumas Nathalie Boilard

Canadian Institutes of Health ResearchBurroughs 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)