Department of Hematology
Balance
Normal
Homeostasis
Control of mRNA translation contributes to the
Regulation of Expansion and Differentiation
of Erythroid Progenitors.
Stress
Erythropoesis
MDS
Godfrey Grech
expansion differentiation
cKit
Epo
activation
levels
Andrzej Nieradka
PI3K/mTOR pathway regulates
translation efficiency
Model system
Epo
SCF
c-Kit
GR
EpoR
PDK1/2
ES
ES
E
E
-
+
-
+
PIP3 PIP3
PH
P-Yp110
5
p8
Epo,
SCF,
glucocorticoids
PTEN
PIP2
PH
PKB
terminal
LY294002 erythroid
differentiation
renewal/proliferation
rheb
Epo
Additional Hits
cKit mutations lead to leukemia
Marieke von Lindern
SCF
Genetic
Hits
TSC1
+SCF
+LY
PI3K
LY
LY294002
+rapa
α –P-4EBP
TSC2
α –4EBP
rapamycine
mTOR
(15μM)
Pi
AUG
AAAAAA
Conclusion 1
• Regulation of translation initiation by eIF4E
is an important pathway stimulated by SCF to
delay
differentiation
of
erythroid
progenitors.
4EBPeIF4E
m7G - Sepharose - m7G
eV
NF
ss
eIF4G
Adapted from Graff JR
& Zimmer SG, Clinical &
Experimental Metastasis
(2003) 20:265-273
Protein synthesis Rate
Ectopic eIF4E promotes
eIF4F complex formation
TOP
eIF4G
eIF4E
m7G
p70S6K
Pi
Pi 4EBP
Pi Pi
4EBP eIF4E
eIF4E
eIF4E
overexpressor
ss
NF
α -eIF4G
α -eIF4E
Blazquez-Domingo et al. MCB Oct 2005; Vol.25 No.19
1
Objectives
Gene expression profiling
polysome bound vs Total mRNA
9 To identify genes that are translationally controlled
upon SCF signaling.
SCF
Hybridise
polysome
bound
RNA
Epo
9 To investigate the contribution of these genes in the
attenuation of erythroid differentiation.
9 To identify regulatory
polysome recruitment
elements
that
Epo+SCF
deprived
ANOVA
Gene List
Hybridise
Total RNA
modulate
Epo+SCF
deprived
Affymetrix
Gene expression profiling
Identification of translational targets
Selection Criteria
cluster analysis
394 genes – ANOVA Gene List (pvalue <0.01)
A (249)
B (89) C
D
Gene List
9
Data Analysis
total RNA
Translationally upregulated
polysome
bound RNA
Down regulated in differentiation
Anova total
N=148
29
5
PI3K
10
10
PTEN
eIF4E
Factor dependent
upregulation
polysome loading
Factor dependent
downregulation
polysome loading
transcription
transcription
signaling
RNA
splicing
transcription
regulation
RNA
stability
Gene Expression
metabolism
post
transcription
modification
ribogenesis
.
prot
processing
ros,
apoptosis
.
RNA
translation
Cell cycle
0
.
.
activate
1. eEF2
2. eIF4B/A
3. rDNA transcription
enhanced
protein synthesis
TOP
mRNAs
eIF4F
sensitive
mRNAs
20
10
0
.
.
post
transcription
modification
Factor dependent
upregulation
Gene Expression
signaling
S6K
10
metabolism
4EBP
PP2A/α4
RNA
splicing
PP2A
20
0
30
transcription
regulation
mTOR
RNA
stability
TSC1
TSC2
0
30
prot
processing
Anova pb/total
N=115
ribogenesis
38
PKB
RNA
translation
1
No of genes
71
20
ros,
apoptosis
269
113
Growth
factor
stimulation
20
Cell cycle
Anova pb
N=458
differentiation
No of genes
eIF4G
eIF4E
.
.
polysome loading
transcription
2
4EBP eIF4E
Pi
Pi 4EBP
Pi Pi
Empty vector
eIF4E
clone
80
60
40
20
Empty vector
ss
NF
E+LY
E+rap
S+LY
S
ES
E
ss
0
NF
ss
NF
E+LY
E+rap
S+LY
S
S+rap
rapamycine
mTOR
0
Fli1 mRNA
100
S+rap
5
p8
PI3K
PKB
PDK1/2
p110
PH
20
ES
PH
P-Y-
40
E
PIP3 PIP3
60
ss
PIP2
Igbp1 a translation controlled mRNA
80
NF
c-Kit
Percent polysome association (%)
SCF
• Is the recruitment to polysomes
sensitive to:
• PI3K
and
mTOR
inhibitors
LY294002 and rapamycine?
• eIF4E overexpression
Percent polysome association (%)
Polysome recruitment of selected
transcripts is PI3K dependent
Polysome recruitment
eIF4E
clone
eIF4E
m7G
TOP
eIF4G
LY294002
AUG
AAAAAA
Functional Analysis of selected
translationally controlled trancripts
Objectives
9 To identify genes that are translationally controlled
upon SCF signaling.
We identified 9 translationally controlled
genes dependent on PI3K/mTOR pathway.
9 To investigate the contribution of these genes in the
attenuation of erythroid differentiation.
9 To identify regulatory
polysome recruitment
elements
that
constitutive expression in erythroid
cell model
modulate
Effect on erythroid differentiation
Constitutive Igbp1 expression blocks
erythroid differentiation
mEd2
mEd2
α4
Igbp1
800
eV
Mean Cell Volume (fl)
5730466P16Rik
5730466P16Rik
Cell number (x 10^6)
100
10
0
24
48
72
Epo time point (hrs)
Kist
Kist
600
400
200
1
Cnih
Cnih
250
Hb per cell vol
Phenotypic Analysis of
Target genes
96
125
0
0
24
48
72
Epo time point (hrs)
96
0
24
48
72
96
Epo time point (hrs)
hnrpa1
hnrpa1
Confocal Microscopy done by Bart Aarts
3
Constitutive Igbp1 expression blocks
erythroid differentiation
800
600
400
0
24
48
72
125
24
48
72
96
Epo time point (hrs)
0
c-Kit
PTEN
24
48
72
0
96
24
48
72
96
Epo time point (hrs)
Epo time point (hrs)
PH
PKB
rheb
PH
P-Yp110
T 72
400
Igbp1
PIP3 PIP3
PDK1/2
PIP2
eV
Igbp1 modulates phosphorylation
of mTOR targets
SCF
EpoR
600
200
0
24
48
72
96
Epo time point (hrs)
Potential role of Igbp1
Epo
10
1
0
0
96
Epo time point (hrs)
Mean Cell Volume (fl)
Igbp1
eV
200
1
800
100
Cell number (x 10^6)
10
250
Hb per cell vol
eV
Igbp1
Mean Cell Volume (fl)
Cell number (x 10^6)
100
Constitutive Igbp1 expression blocks
erythroid differentiation
TSC1
constitutive Igbp1 expression
empty vector
5
p8
TSC2
ss
PI3K
mTOR
dep Epo SCF
ES
ss
dep Epo SCF
ES
S6K-P
S6K
Epo
Pi
Pi
p70S6K
p70S6K
4EBP
Pi 4EBP
Pi Pi
Igbp1
Epo
SCF
ss dep 10’ 60’ 10’
SCF
ss dep 10’ 60’ 10’
60’
60’
4EBP
pp2a
TOP
AUG
AAAAAA
Igbp1 modulates phosphorylation
of mTOR targets
Epo
c-Kit
PTEN
PIP3 PIP3
PIP3 PIP3
5
p8
PH
PH
P-Y-
rheb
TSC1
PIP2
PKB
p110
PTEN
EpoR
PDK1/2
PH
P-Y-
p110
5
p8
PH
PKB
PIP2
PDK1/2
EpoR
TSC2
PI3K
mTOR
Pi
p70S6K
TSC1
TSC2
PI3K
p70S6K
4EBP
mTOR
Pi
Pi 4EBP
Pi Pi
Pi
p70S6K
p70S6K
4EBP
Pi
Pi 4EBP
Pi Pi
Igbp1
pp2a
pp2a
eIF4E
m7G
TOP
Igbp1
pp2a
AUG
AAAAAA
m7G
TOP
eIF4G
c-Kit
Igbp1 modulates phosphorylation
of mTOR targets
Epo
rheb
eIF4E
m7G
eIF4G
pp2a
AUG
AAAAAA
4
mEd2 inhibits
erythroid differentiation
Cellular
Stress
UV,
Proteosome
inhibition,
ER
low nutrients stress
Epo (differentiation) conditions
t0
72hrs
low heme,
heat shock,
oxidative Viral
stress
infection
PEK HRI
Re-establish
eIF2α activity
(feedback)
GCN2
PKR
eIF2α
eIF2-P
enhanced ATF6
eIF2B-GTP
expression of mEd2
wt
exchange factor
Constitutive
activation
of alpha4
eIF2B-GDP
Dephosphorylation of
eIF2α
eIF2-GTP
Constitutive
activation
of mEd2 (C-terminus)
eIF2 α
sensitive
mRNAs
Conclusions
•
Like SCF signaling, Igbp1 enhances translation
initiation efficiency by modulating phosphorylation
status of mTOR targets. This results in a block of
erythroid differentiation.
9 To investigate the contribution of these genes in the
attenuation of erythroid differentiation.
9 To identify regulatory
polysome recruitment
elements
that
modulate
Regulatory elements within UTR Structures
eIF4E
easy
5’
3’
difficult
eIF4E
eIF4E
eIF4E
5’
eIF4E
eIF4E
eIF4G
Constitutive expression of Igbp1 results in
hyperphosphorylation of 4EBP and S6K
phosphorylation upon Epo stimulation.
eIF4G
•
9 To identify genes that are translationally controlled
upon SCF signaling.
eIF4G
Constitutive expression of Igbp1 blocks erythroid
differentiation.
eIF4G
•
Objectives
eIF4G
We identified a list of translationally controlled
genes dependent on PI3K/mTOR pathway
eIF4G
•
efficient lysosomal
degradation
3’
very difficult
5’
3’
5
Regulatory elements within UTR uORF
Identify regulatory sequences
• Identify Transcription Start Site (full length
UTR) using RACE
eIF4G
• Bioinformatics:
eIF4E
uAUGt
AnnAUGG
• Invitro transcription/translation to screen
putative elements
eIF4E
eIF4G
eIF4G
eIF4E
• to scan for known elements
• RNA secondary structures
stop
uAUG
AUG
• Mutate elements to assess contribution in
translation efficiency
Re-initiation
Identify regulatory sequences
• Identify Transcription Start Site (full length
UTR) using RACE
Sequencing of RACE products
– Igbp1
• Bioinformatics:
• to scan for known elements
• RNA secondary structures
• Invitro transcription/translation to screen
putative elements
• Mutate elements to assess contribution in
translation efficiency
Sequencing of RACE products
– mEd2
Sequencing of RACE products
– Nm23
A
GGCCAGGTA (Nx)AAG
GGCCAG
ATGG
ATGG
with intron
without intron
201bp
NCBI_Nm23
439bp
EST_Nm23
483bp
Race_Nm23
B
AUG
6
Identify regulatory sequences
GAIT- Gamma interferon activated inhibitor of
Ceruloplasmin mRNA translation
• Identify Transcription Start Site (full length
UTR) using RACE
• Bioinformatics:
• to scan for known elements
• RNA secondary structures
• Invitro transcription/translation to screen
putative elements
• Mutate elements to assess contribution in
translation efficiency
UTR sequencies recognised as GAIT
elements – Are they GAITs?
The GAIT structure has a striking similarity to the Iron
Responsive Element (IRE) which is also involved in RNA/protein
association.
Such structures recruit protein complexes in response to
iron (IRE) and interferon gamma (GAIT). Elements with common
features suggest sites of protein binding that might be responsive
to signaling.
GAIT
SCHEME
Nm23 - structure
Identify regulatory sequences
• Identify Transcription Start Site (full length
UTR) using RACE
• Bioinformatics:
• to scan for known elements
• RNA secondary structures
• Invitro transcription/translation to screen
putative elements
• Mutate elements to assess contribution in
translation efficiency
Disruption of regulatory sequences
A
-306
Nm23_5’UTR
-217
TCCCTCCTCTCTCC
*
IR
IR
-3
AccATGg
M
AnnAUGg....tgaAUGcagAUG.....gait1..tga......gait2...CGGGU
oligo pyrimidine tract
Nm23 genomic - transcript alignment
GGUA
mEd2_5’UTR
-21.30 kcal / mol
B
-20 kcal/mol
-263
TCCCTCCTCTCTCC
IR
-217
IR
*
-3
AccATGg
M
NF61
NF61 proviral integration flanked by
.
7
B
Pro-viral insertion – effect on gene expression
-263
TCCCTCCTCTCTCC
IR
-217
IR
-3
AccATGg
M
NF61 proviral integration flanked by
subpolysomal
C
A. Gene activation by promoter insertion
.
+
polysome
bound
B. Gene activation by enhancement or
removal of mRNA (de)stabilizing motifs
+
EtBr
NF
22%
IL3
66%
NF
61%
IL3
73%
NFS 36
NFS 61
Identify regulatory sequences
• Identify Transcription Start Site (full length
UTR) using RACE
• Bioinformatics:
• to scan for known elements
C. Gene disruption
D. UTR disruption
Novel retroviral regulation
Invitro transcription/translation to screen
putative
elements
in vitro
A
400
G1
G2
200
Nm23 full length UTR
G=Gait element
uORFs deleted
0
30
• RNA secondary structures
6
• Invitro transcription/translation to screen
putative elements
• Mutate elements to assess contribution in
translation efficiency
Nm23-M2
AUG
60
4
AUG
2
mEd2 uORFs deleted
0
30
60
90
time (minutes)
full length
Identify regulatory sequences
• Identify Transcription Start Site (full length
UTR) using RACE
• Bioinformatics:
HD3
BA/F3
100
100
1.5
1.5
80
80
1
60
0.5
• to scan for known elements
60
• RNA secondary structures
40
40
20
20
0.5
ss
• Mutate elements to assess contribution in
translation efficiency
del uAUGs
Mutate elements to assess contribution in
translation efficiency
1
• Invitro transcription/translation to screen
putative elements
90
mEd2
NF
0
0
ss
NF
Nm23-M2 full length
ss
NF
Nm23-M2 uORFs deleted
8
Current Experiments
• Characterise pro-viral insertions within the UTR
of Nm23 using tumours derived from retroviral
insertion mutagenesis.
• Mutation analysis on Nm23 UTRs to disrupt
putative protein-binding elements and uORF
(T7).
• Optimisation of RACE experiments for Igbp1
UTR determination.
• Identify regulatory elements
translationally controlled genes.
in
list
of
9