Paper presentation Lucas Kemper 06.12.2010
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Transcript of Paper presentation Lucas Kemper 06.12.2010
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Paper presentation
Lucas Kemper
06.12.2010
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Introduction
microRNA
Lindsay, A.M.; Cell, 2008
transport of pre-miRNAby Exportin 5
block of mRNA translationhigher mRNA instability
mRNA cleavage
inducible expressionof pri-miRNA withhairpin structure
cleavage by Droshain the nucleus
cleavage by Dicerin the cytoplasm
miRISC binding to5‘or 3‘ UTR of mRNA
formation of themiRNA-induced
silencing complex
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Introduction
KSHV
Kaposi‘s sarcoma
Kaposi‘s sarcoma-associated herpesvirus
also known as human herpesvirus 8 (HHA8)
structure
can induce several human cancers
LEC
primary lymphatic endothelial cells
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Aim
determine the role of host microRNAs in the innate immune response
during virus infection
innate immune response
uninfectedcell
infectedcell
antiviral state
microRNA
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Effect of KSHV infection on the miRNA profile of LECs
microarrayqRT-PCR
several host miRNAs upregulated by KSHV infection identification of two groups of upregulated miRNA (early / late)
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Testing potential role of identified miRNAs in the antiviral response
2d
aft
er
infe
ctio
n
inhibition of two early miR suppresses viral gene expression inhibiting late miR has no effect on viral gene expression
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miR-132 miR-146a
How to continue?
vs.
Implication in neuronal differentiation and function
Induced after LPS-stimulation of monocytic cell lines
Overexpressed in chronic lymphoblastic leukaemia
primary miRtranscript
miR-132
miR-212 less stableweaker inductionlower effect
regulated by CREB
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fold
incr
ease
to u
ninf
ecte
d
miR-132 induction is a result of viral bindingand not of viral gene expression
miR-132 induction is fast an transient
miR-132 induction based on viral binding
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miR-132 is induced through a CREB-dependent mechanism
kinetics of CREBphosphorylation
chemical inhibitionof MAPKs
siRNA knock downof CREB & ERK1/2
CREB phosphorylationdependent on MAPKs
induction of miR-132 mediated by ERK-CREB dependent pathway
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p300 is a target of miR-132
Using bioinformatics:
PITA TargetScan EiMMO Miranda
Search algorithms to predict bindingbetween microRNAs and mRNAs
p300 is a putative target of miR-132
miR-132 targets an 8mer site in the UTR of p300
co-activator
essential for initiation of antiviral immunity
interacts with CREB
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p300 is a target of miR-132
LEC
s
transfected contructs
LEC
s
miR-132 reduces p300 protein level which is functionally relevant
transfected contructs
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p300 is a target of miR-132
6h p.i.
fold
incr
ease
to u
ninf
ecte
d
miR-132 induces its own feedback loop
feedback loop explains partly transient miR-132 induction
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miR-132 inhibits the antiviral interferon responseand enhances viral replication
inhibition of miR-132 leads to
less KSHV replication
more IFN-β and ISG expression
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miR-132 regulates viral gene expressionthrough suppression of p300
p300 protein level is reduced due to miR-132
KSHV
viral mRNA level is increased due to miR-132
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Summary
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This work reveals:
a viral gene expression-independent, host-microRNA-mediated mechanism that regulates the antiviral response.
Summary
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Thank you
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…..
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RenillaUTR
HeLa
+wt or mutated
miR132_212 cluster
miR-132_212 cluster represses a construct containing p300 UTR
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miR-132 induced repression of p300 affects p300 regulated gene expression
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miR-132 regulates viral gene expressionthrough suppression of p300
viral
gene
s
knock down of p300 reduces antiviral resonse after KSHV infection
knock down of p300 promotes viral gene expression