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

Seite 2 |

• 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

Seite 3 |

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

Seite 4 |

Depending on trans-acting factors binding to cis-

regulatory elements

microRNAs (miRNAs)

RNA-binding proteins

Coope-ration

&exclusion

Open questions

Seite 5 |

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

Seite 7 |

ICOS expression is controlled by roquin

Seite 8 |

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

Seite 9 |

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

Seite 10 |

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

Seite 13 |

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

Seite 14 |

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

Seite 16 |

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

Seite 17 |

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

Seite 18 |

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

Seite 19 |

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

Seite 20 |

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

Seite 21 |

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

Seite 23 |

http://www.biochem.arizona.edu

Binding position of roquin to ICOS mRNA

Seite 24 |

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

Seite 25 |

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.

Seite 27 |