blast search (330-nt 3’utr element) - 3'utr of ~47 l. major orfs (65-96% identity) 5'...
Post on 19-Dec-2015
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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//
//
//
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)