dies ist nur ein beispielbild! roquin binds inducible costimulator (icos) mrna and effectors of mrna...

Dies ist nur ein Beispielbild!Roquin binds inducible costimulator (ICOS) mRNA and

effectors of mRNA decay to induce microRNA-independent

post-transcriptional repression

Glasmacher et al., Nat Immunol. 2010 Aug;11(8):725-33

Kirstin Jurrat

Paper Presentation, 1st November 2010

ZIB

Storybook

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• Who is who• Degradation of mRNA

Introduction

• Repression• Essential roquin domains

Roquin controls ICOS mRNA

• P-body pathway• Role of stress granules• Decapping proteins• miRNA & Dicer & RISC

Details of repression

• Involved roquin domains• Binding position at ICOS mRNA

Interaction with ICOS mRNA

Summary & Discussion

Introduction – Who is who

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Inducible costimulator (ICOS)

• On surface of T-cells after antigen recognition

• Post-transcriptional regulation assumed

• Effect: regulation of cytokine production provides B-cells for

antibody production

Relationship of ICOS and roquin

• Roquin: CCCH-type zinc finger protein, destabilizes mRNA of

ICOS

• Loss of repression leads to autoimmunity (Rc3h1san/san mice)

Post-transcriptional regulation of gene expression

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Depending on trans-acting factors binding to cis-

regulatory elements

microRNAs (miRNAs)

RNA-binding proteins

Coope-ration

&exclusion

Open questions

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ICOS repression by roquin

• Roquin: destabilizes mRNA of inducible costimulator (ICOS)

• Required: 3´untranslated region (3´UTR) of ICOS mRNA with putative

miRNA-binding site possible functional dependence of roquin on

miRNA

• miRNA

• Roquin-miRNA

• Roquin-miRNA-induced silencing complex

Questions

• how ICOS mRNA is recognized by roquin

• how recognition induces mRNA decay

mRNA degradation

Degradation of eukaryotic mRNA: Decapping & P-bodies

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ICOS expression is controlled by roquin

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In TH cells: Expression of ICOS under post-

transcriptional control of roquin

- Primary CD4+ T cells from Tg(DO11.10)-Tg(CAR∆-1) mice: permissive

for adenoviral infection

- Adenoviral infection and activation with anti-CD3 and antiCD28

Roquin domains needed for ICOS repression

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Roquin (1-951): slightly less active than WT roquin

Roquin (1-509): inactivation of roquin-mediated ICOS repression

Superinfection of mouse embryonic fibroblasts (MEFs) with retrovirus

containing Thy-1.1 plus WT or mutant roquin

Roquin interacts with the P-body pathway

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No Stress

FMRP diffuse located no stress granulaGFP-roquin shows full colocalization with P-bodies

RNA-heli

case

P-b

odies

mar

ker p

rot.

for

stres

s gra

nules

Roquin

Overla

y

Roquin interacts with the P-body pathway

RNA-heli

case

P-b

odies

mar

ker p

rot.

for

stres

s gra

nules

Roquin

Overla

y

After stress induction

Association with stress granules

Lost connection to P-bodies

Roquin interacts with the P-body pathway

lost focus in absence of stress

still able to colocalize with stress granula after stress induction

GFP-roquin (1-749): missing Gln and Asn enriched sequences

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Expression of ICOS and

roquin in NIH3T3 cells

Roquin interacts with the P-body pathway

Roquin (1-749) impaired

of ICOS downregulation

Roquin interaction with P-body pathway independent of

stress granules?

Confirms correlation of roquin function with localization to P-bodies

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Roquin interaction with P-body pathway independent of stress granules?

Expression of ICOS and

roquin in WT and TIA-1

deficient MEFs

Roquin mediated repression of ICOS does not depend on Tia-1

function in absence of stress

Looking for interaction partners: Co-Immunoprecipitation

Antigen binds to interaction partner

Protein A/G binds antibody

Antibody binds antigen

http://de.academic.ru

Roquin interacts with decapping proteins

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Insensitive to RNase treatment

mRNA independent

• Rck (essential for

decapping)

• Edc4 (decapping enhancer)

• Dcp1a (decapping activator)

Co-Immunoprecipitation of

roquin

Roquin interacts with decapping proteins

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Model: roquin mediates silencing of ICOS through functional

and physical interaction with proteins of the decapping

pathway

Knockdown of Rck ICOS expression increased

Roquin-mediated repression does not require miRNA

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Rck also essential in miRNA

dependent post transcriptional

signaling

influence of miRNA on roquin-

mediated ICOS downregulation

to be checked

Roquin-mediated repression does not require miRNA & Dicer

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Experiment

- MEFs, WT and deficient in

endoribonuclease Dicer1 (Dicer1 fl/fl)

- retroviruses: ICOS & WT

roquin/roquin (1-509)

Results:

WT roquin downregulated ICOS

mRNA in WT and Dicer-deficient

cells

downregulation similar ratesroquin induced similar decrease of ICOS mRNA expression

in WT and Dicer-deficient cells

Roquin-mediated repression does not require miRNA & Dicer

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roquin down-

regulation of ICOS mRNA is

Dicer independent

ICOS and Hoxc8 mRNA expression measured

MEFs (WT &

Dicer

deficient)

Roquin-mediated repression does not require RISC

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Embryonic stem cells

B9: Ago1-, Ago3-, Ago4-

E7: Ago1-Ago4-, transgenic Ago2

adenoviral coinfection of ICOS 3´UTR with GFP-CTRL or GFP-roquin

Roquin dependent ICOS repression independent of central RISC components

Roquin is an RNA-binding protein

Immunoprecipitation of Roquin shows

Roquin is directly binding to ICOS mRNA

binding via ROQ domain in collaboration with CCCH-

type zinc finger

Electrophoretic mobility shift assay

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http://www.biochem.arizona.edu

Binding position of roquin to ICOS mRNA

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Electrophoretic mobility shift assay

roquin interacts with Full-length ICOS mRNA

indicates several binding sites of roquin at ICOS mRNA

3´UTR at ICOS mRNA essential for roquin binding

significant region between 100 and 200 basepairs 3´of stop codon

the longer the 3´UTR the stronger is repression by roquin

further indication for more than one binding site at 3´UTR

Summary & Discussion

- in TH cells: expression of ICOS under post-transcriptional control of

roquin

- roquin (1-509): inactivation of roquin-mediated ICOS repression

- roquin interacts with the P-body pathway via Gln/Asn rich regions

(roquin (1-749)) and independent of stress granules

- roquin interacts with decapping proteins independent of mRNA

- roquin-mediated repression does not require miRNA, Dicer and

RISC complex

- roquin binds with N-term. directly to 3´UTR of ICOS mRNA at

several binding position

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Thank you for your attention!

Literature

Presentation based on:

Roquin binds inducible costimulator mRNA and effectors of mRNA decay to induce microRNA-

independent post-transcriptional repression.

Glasmacher E, Hoefig KP, Vogel KU, Rath N, Du L, Wolf C, Kremmer E, Wang X, Heissmeyer V.

Nat Immunol. 2010 Aug;11(8):725-33. Epub 2010 Jul 18.

Further literature:

Polysomes, P bodies and Stress granules: States and Fates of Eukaryotic mRNAs

Vidya Balagopal and Roy Parker*

Curr Opin Cell Biol. 2009 June ; 21(3): 403–408. doi:10.1016/j.ceb.2009.03.005.

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