edu

1. Post-transcriptional regulation of mRNAs containing A+U-rich elements by the VHL tumor suppressor
James Gnarra, Julie Brown, and Weijun Liu
Department of Urology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15232
1. VHL-, oxygen-, and proteasome-dependent regulation of
AUF1 expression in RCC and 293T cells
786-O G7F
- 50 kDa H.C.
AUF1 | - 41 kDa
- 17
- 75 kDa
A, pVHL-positive 786-O G7F RCC cells; B, WT7 RCC cells; or C, 293T cells were extracted with Igepal lysis buffer,
and immunoprecipitations were performed using 500 g cellular protein and antibodies directed against AUF1 or pVHL
(Ig32 for 293T cells or Flag for 786-O G7F RCC cells) or irrelevant mouse or rabbit IgG (mIgG or rIgG).
- 50 kDa
- 50 kDa
- 37
- 75 kDa
Cul2
- 32
- 41
2
1
E 20
F
18
16
14
12
10
8
6
4
2
AUF1 levels in pVHL-positive (786-O G7F) and pVHL-negative (786-O 157 RCC cells that were cultured (A) under normoxic (room
air) or hypoxic (1% O2) conditions for 18 h or (B) in the presence of the hypoxia mimetic, desferrioxamine (DFO; 100 M), for 2 to 18
h. U, untreated cells. C, 293T cells were cultured under normoxic or hypoxic conditions for 18 h. D, 786-O G7F RCC cells were
cultured in the absence or the presence of the proteasome inhibitor, MG132 (20 M), for 2 h. E, Quantitative real-time RT-PCR
analysis of VEGFA and AUF1 mRNAs in 293T cells that were cultured in normoxia or hypoxia for 18 h. F, Relative AUF1 mRNA levels
in pVHL-positive, WT7, and pVHL-negative, PRC9, RCC cells were determined by quantitative real-time RT-PCR.
5. pVHL-dependent ubiquitylation of p42 & p45 AUF1
AUF1 Blot HA Blot
Ub-AUF1
p25 VHL-myc - 31 kDa
p45
123 -
86 -
44 -
32 -
123 -
86 -
A, UOK121 RCC cells were transfected with plasmids expressing Myc-tagged pVHL, HA-tagged ubiquitin, or both.
MG132 (10 M) was added 24 h post-transfection, and after an additional 8 h, anti-myc immunoprecipitations were
performed. Western blotting was performed with AUF1 and HA antibodies. Blots were stripped and re-probed for pVHL
(bottom). B, p25VHL, p42AUF1, and p45AUF1 were synthesized in vitro in separate reactions. Programmed reticulocyte
lysate were mixed and incubated with recombinant E1 ubiquitin-activating enzyme and UbcH5b E2 ubiquitin-conjugating
enzyme, ubiquitin, ubiquitin aldehyde, and an ATP regeneration system at 37°C for 4 hours. Western blots blots were
probed first with anti-AUF1 (top) and then stripped and re-probed with anti-ubiquitin (bottom). p37 and p40 AUF1 are not
targeted for ubiquitylation by pVHL (data not shown).
6. AUF1 and HuR associate with VEGFA mRNA in
vivo and regulate VEGFA mRNA levels
pVHL
(GXEEX)8 b domain a domain b
4. Opposing effects of AUF1 and HuR on VEGFA mRNA
AUF1 knockdown resulted in increased VEGFA mRNA levels
HuR knockdown resulted in decreased VEGFA mRNA levels
Silencing: AUF1 HuR
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
293T cells were transfected with a vector expressing AUF1 shRNA, an HuR siRNA pool, or
appropriate controls. Total RNA was extracted 48 h post-transfection and quantitative real-time
RT-PCR analyses were performed using VEGF-, AUF1, or HuR-specific PCR primers.
Expression levels of the target mRNAs were determined in triplicate and averages are
expressed relative to TBP mRNA levels, which were unaffected in these experiments.
HRE HRE
B. The pVHL E3 Ubiquitin Ligase Complex
exon 2 19 aa
AUF1 p40 + p45
QYQQQQQ
RRM1 RRM2 AUF1 p37
exon 7 49 aa
PSQNWNQGYSNYWNQGYGNYGYNSQGYGGYGGYDYTGYNNYYGYGDYSN
AUF1 p42 + p45
Normoxia VHL
AUF1 HuR
5’ UTR AU-Rich Elements
Hypoxia
AUF1 HuR
b
a
b
a
Funding by NCI CA78335 and CA125930
2. Co-immunoprecipitation of pVHL and AUF1 3. In vitro interaction domain mapping of pVHL and
AUF1
A B
U 2 4 8 18 U 2 4 8 18 DFO, hrs
- 41 kDa
- 32
AUF1 |
786-O G7F 786-O 157D
- 50 Tubulin - 41
AUF1 |
HuR
Tubulin
- 37
- 37
Normoxia Hypoxia
C
AUF1 |
293 cells
- 50
D
- 41 kDa
Tubulin
786-O G7F
AUF1 |
WT7 PRC9
0
Relative AAUF1 mRNA
AUF1
0
RQ
VEGF
293T
N 6 16 N 6 16 h, hypoxia
p25 VHL 
p19 VHL 
- 30 kDa
- 17
AUF1 |
- 50 kDa
- 37
Cul2 
- 75 kDa
- 50
WT7
AUF1 |
- 37
HA-VHL 
- 50 kDa
H.C.
- 37
- 25
A B
- 32
p19 VHL 
293
Cul2 
- 50
C
 Actin
60
50
40
30
20
10
bp 600 -
400 -
200 -
600 -
786-O G7F 786-O 157D
786-O 157D
B
0
RQ
A
Hypoxia - + - +
VEGF
78 89 101 114 173 213
1 54
AUF1
RRM1 RRM2 Q
VHL
weak
VHL
strong
p45 AUF1
112
|
285
|
355
|
1
|
78
|
Reciprocal GST pull-down studies showed that p45AUF1 interacted with amino acids 1-89 of p25VHL
and p25VHL interacted strongly with p45AUF1 amino acids 285-355 and weakly with amino acids 78-112
A. The focus of our work has been on post-transcriptional mechanisms
of regulation of VEGFA expression
Post-Transcriptional
mRNA stability
Transcriptional
AU-Rich Elements
AREs
1 kb
5’ UTR
HIF SP1
alternative splicing
HRE
STAT (AUUUA)
Proteasome Degradation
C. Four AUF1 RNA binding proteins are expressed through
alternative splicing
VHL-myc + + - - + +
HA-Ub + - - - - +
- 123
- 83
- 41
- 31
AUF1 |
pVHL Blot
A
H.C.
1 2 3 4 5 6
pVHL
E2
AUF1 p42
AUF1 p45
+
-
+
-
-
+
-
+
-
+
+
-
+
-
-
+
+
+
-
+
+
+
+
-
p42
AUF1 blot
ubiquitin blot
44 -
32 -
B
1 2 3 4 5 6
L.C.
UOK121
AUF1--many activities
•Binds A+U-rich sequences
•Binds telomere repeat sequences
•Transcriptional regulator in association with nucleolin
D. Model for transcriptional and post-transcriptional regulation of
VEGFA expression in normoxia and in hypoxia
0
Relative mRNA Levels
VEGF
AUF1
HuR
VEGF
UGA
control ARE1
VEGF 3’ UTR
A
113 305 498
Luc-ARE1 Luc-control
N H N H
Luciferase PCR
Actin PCR
B
bp 800 -
600 -
600 -
D
Luc-ARE1
Normoxia
Luc-ARE1
Hypoxia
Luc-control
Normoxia
C ip:
bp 800 -
600 -
800 -
600 -
600 -
A, pVHL-positive RCC cells (786-O G7F) and pVHL-negative RCC cells (786-O 157D) were cultured
under normoxic conditions (room air) or hypoxic conditions (1% O2) for 18 hours. Total cellular RNA was
prepared and quantitative real time RT-PCR reactions were performed with VEGFA-specific PCR primers.
B, RNP-immunoprecipitations were performed from 786-O 157D RCC cells with antibodies directed against
AUF1, HuR, or pVHL, or an irrelevant rabbit IgG (rIgG). Immunoprecipitates were washed in lysis buffer,
RNA was extracted, and RT-PCR was performed using VEGF primers that amplify alternatively spliced
mRNAs or actin PCR primers that amplify a 600 bp fragment.
7. Oxygen-dependent and pVHL-dependent expression of
luciferase-VEGFA ARE1 constructs
A, A diagram of the proximal portion of the VEGF 3’ UTR showing the regions that were independently subcloned
into the 3’ UTR of the luciferase reporter. B, 293T cells were transiently transfected with pGL3-Promoter
luciferase reporter plasmid containing VEGFA ARE1 (Luc-ARE1) or VEGF control sequences (Luc-control), and
cells were cultured under normoxic (room air) or hypoxic (1% O2) for 18h. Total RNA was extracted and RT-PCR
using luciferase or actin PCR primers was performed. C, Luc-ARE1 mRNA half-life was determined in transfected
293T cells. D, 293T cells were transfected with Luc-ARE1, and RNP-immunoprecipitations were performed with
pVHL, AUF1, HuR, or irrelevant rabbit IgG (rIgG). Immunoprecipitates were washed, RNA was extracted, and
RT-PCR was performed using luciferase PCR primers.
5’ UTR AU-Rich Elements
b
a
Protein ubiquitylation
& degradation
RNA Degradation
HIF