Transcript p300

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