1. The regulation of microRNA activity by ATM in the DNA damage response
David W. Salzman1, Frank Slack2, Joanne B. Weidhaas1
1Department of Therapeutic Radiology, Yale University School of Medicine
2Department of Molecular, Cellular and Developmental Biology, Yale University
MicroRNAs are a class of non-coding, 18-24nucleotide, trans-acting RNAs
that negatively regulate gene expression at the posttranscriptional level by
annealing to cis-regulatory element(s) in a target mRNA and inhibiting
protein translation. The temporal and spatial expression of microRNAs
proceeds through several regulatory checkpoints mainly, transcription and
processing of the pri-microRNA and pre-microRNA precursors by the
RNaseIII enzymes Drosha and Dicer.
Previous work indicates that miR-34 is regulated at the transcriptional level
following DNA damage in a P53-dependent manor and protects cells
against DNA damage-induced apoptosis by targeting a number of critical
genes required for cell cycle progression. However, data from several
laboratories (including our own) suggests that there is a pool of
ubiquitously expressed miR-34 in cells, the expression of which is
independent of both P53 and DNA damage.
The role of P53/DNA damage-independent miR-34 is not understood. To
study this, we established a luciferase reporter assay to measure miR-34
gene silencing activity. Our data indicated that the pre-existing pool of
miR-34 is inactive (not competent to silence gene expression). However,
the pool of miR-34 was rapidly activated by ionizing radiation (IR) which
occurred several hours before P53-mediated transcription.
Introduction
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let-7 miR-34
microRNAactivity
(folddifferenceMT/WT)
MCF-7
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let-7 miR-34
microRNAactivity
(folddifferenceMT/WT)
H460
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let-7 miR-34
microRNAactivity
(folddifferenceMT/WT)
A549
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let-7 miR-34
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Figure 1: Can we measure miR-34 activity?
Hypothesis
WT target CGACCGUCACAGAAUCGACCAACAGA
||||||||||||||||||||||
miR-34 UGGCAGUGUCUUAGCUGGUUGU
| |||| |||||||||||
MT target CGAGGCACACACUAUCGACCAACAGA
A. B.
Transfect:
WT or MT reporter
Measure dual-luciferase
miRNA activity = fold suppression of MT/WT
C.
D. The pool of inactive miR-34 is rapidly
stimulated by radiation before
transcription. A. Sequence of the wild-
type (WT) and mutant (MT) miR-34 target
element subcloned into the 3’UTR of the
renilla luciferase mRNA in the
psiCHECK2 dual luciferase reporter. B.
Schematic diagram of how experiments
are preformed. C. The level of let-7 and
miR-34 gene silencing activity in A549,
HeLa, MCF-7 and H460 cells, D.
Simultaneous measurement of miR-34
activity and expression in A549 cells +/-
ionizing radiation.
miR-34 activity is regulated at the level of the mature microRNA
Figure 2: Is IR-induced miR-34 activity Dicer dependent?
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No siRNA siP53 siDROSHA siDICER siAGO2 siGAPD
pri-microRNAexpression
(foldchange)
pri-miR-34a pri-miR-34b/c pri-miR-17
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No siRNA siP53 siDROSHA siDICER siAGO2 siGAPD
microRNAexpression
(foldchange)
miR-34a miR-34b miR-34c miR-17
0
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No siRNA siP53 siDROSHA siDICER siAGO2 siGAPD
miR-34activity
-IR +IR
Pre-treat cells:
siRNA-P53
siRNA-Drosha
siRNA-Dicer
siRNA-Ago2
siRNA-GAPD
Transfect with:
WT and MT miR-34 reporter
A. A549
-/+ IR
pri-miR-34
RT-qPCR
Norm. to B-Actin
miR-34
RT-qPCR
Norm. to U6
miR-34
activity
western blot
Norm. to Vimentin
B.
E.
VIM
Drosha
P53
_ +
_
+
_
+
_
+
Vim
Dicer
Ago2
IR
GAPD
_ +
_
+
_
+ IR
IR-induced miR-34 activity is Drosha and Dicer-independent. A. Schematic diagram of the
experiment. A549 cells pre-treated with siRNA for 36-hours were transfected with either the WT or
MT miR-34 luciferase reporters. Cells were exposed to 4Gy of ionizing radiation. Following a 30-
hour incubation the cells were lysed and assayed for pri-miR-34, mature miR-34, miR-34 activity and
protein to validate siRNA knockdown. B. Relative pri-miR-34 and pri-miR-17 (negative control)
expression in irradiated cells. C. Relative mature miR-34 or miR-17 (negative control) expression in
irradiated cells. D. Relative miR-34 activity in irradiated cells. *Graphed for each experiment is the
mean and standard deviation of 2-independent experiments preformed in triplicate. E. Protein
knockdown was validated by western blot.
C.
D.
Summary
1. There is a pre-existing pool of miR-34 in cells
2. miR-34 is stimulated by DNA damage in a Dicer-independent
manor
3. The pre-existing pool of miR-34 is not loaded into Ago2
4. ATM is required for early activation of miR-34, whereas P53 is
required for late activation
5. ATM is required for 5’-end phosphorylation of miR-34
6. We have discovered a novel mechanism for the regulation of
microRNA activity
7. Our hypothesis is true: miR-34 is regulated at the mature level
Figure 4: What DDR pathway activates miR-34?
Pre-treat cells:
siRNA-ATR
siRNA-ATM
siRNA-P53
Transfect with:
WT and MT miR-34 reporter
A549
-/+ IR
miR-34
RT-qPCR
Norm. to U6
miR-34
activity 0
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no siRNA ATR siRNA ATM siRNA P53 siRNA
RelativemiR-34activity
0hr 4hr 12hr 36hr
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no siRNA ATR siRNA ATM siRNA P53 siRNA
RelativemiR-34aexpression
0hr 4hr 12hr 36hr
B.A.
ATM is required for early miR-34 activation, whereas P53 is required for late miR-34
activation. A. Schematic diagram of the experiment. A549 cells pre-treated with siRNA for 36-hours
were transfected with either the WT or MT miR-34 luciferase reporters. Cells were exposed to 4Gy
of ionizing radiation. Following a 30-hour incubation the cells were lysed and assayed for mature
miR-34 and miR-34 activity. B. miR-34 expression in irradiated cells treated with siRNA. C. Relative
miR-34 activity in irradiated cells treated with siRNA.
C.
Figure 3: Is miR-34 loaded into Ago2?
Measure miR-34 by RT-qPCR
A549
FLAG/HA-EGFP
FLAG/HA-AGO2
-/+ IR
FLAGTotal
A.
miR-34 is loaded into Ago2 following IR. A. Schematic outline of the experiment. A549 cells
stably expressing FLAG/HA-Ago2 or FLAG/HA-EGFP (negative control) were exposed to 4Gy of
ionizing radiation. The cells were lysed at the indicated time. miR-34 expression was analyzed in the
total extract or in FLAG immunoprecipitates. B. Relative miR-34 expression in total cell extract (top)
and FLAG immunoprecipitates (bottom).
B.
Acknoledgements
This work was funded by: NIH/NCI 5 K08 CA124484-05 and a Kalimeris Seed Grant.
Figure 5: ATM is required for miR-34 5’-end phosphorylation
miR-34 Northern blot
A549
No siRNA
siRNA-ATM
-/+ IR
Extract RNA
-/+ CIP
A. B.
ATM is required for miR-34 5’-end phosphorylation. A. Schematic diagram of the experiment.
A549 cells pre-treated with siRNA directed against ATM were exposed to 2Gy of ionizing radiation.
Cells were lysed at the indicated time and total RNA was extracted. RNA was treated and untreated
with CIP (to remove 5’-phosphates). miR-34 was analyzed by northern blot. B. Northern blot
analysis of miR-34. 18S rRNA was used to normalize.