1. Kirstin Jurrat Paper Presentation, 1st November 2010
Roquin binds inducible costimulator (ICOS) mRNA and effectors of mRNA decay to induce microRNA-independent post-transcriptional repression Glasmacher et al., Nat Immunol Aug;11(8):725-33
Kirstin Jurrat
Paper Presentation, 1st November 2010

2. Storybook Introduction Roquin controls ICOS mRNA Details of repression
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

3. Introduction – Who is who
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)

4. Post-transcriptional regulation of gene expression
Depending on trans-acting factors binding to cis-regulatory elements
microRNAs (miRNAs)
RNA-binding proteins
Coope-ration
&
exclusion

5. Open questions ICOS repression by roquin Questions
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

6. mRNA degradation

7. Degradation of eukaryotic mRNA: Decapping & P-bodies

8. ICOS expression is controlled by 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
 In TH cells: Expression of ICOS under post-transcriptional control of roquin

9. Roquin domains needed for ICOS repression
Superinfection of mouse embryonic fibroblasts (MEFs) with retrovirus containing Thy-1.1 plus WT or mutant roquin
 Roquin (1-951): slightly less active than WT roquin
 Roquin (1-509): inactivation of roquin-mediated ICOS repression

10. Roquin interacts with the P-body pathway
RNA-helicase
 P-bodies
marker prot. for stress granules
Roquin
Overlay
No Stress
FMRP diffuse located  no stress granula
GFP-roquin shows full colocalization with P-bodies

11. Roquin interacts with the P-body pathway
RNA-helicase
 P-bodies
marker prot. for stress granules
Roquin
Overlay
After stress induction
Association with stress granules
Lost connection to P-bodies

12. Roquin interacts with the P-body pathway
 GFP-roquin (1-749): missing Gln and Asn enriched sequences
lost focus in absence of stress
still able to colocalize with stress granula after stress induction

13. Roquin interaction with P-body pathway independent of stress granules?
Roquin interacts with the P-body pathway
Expression of ICOS and roquin in NIH3T3 cells
Roquin (1-749) impaired of ICOS downregulation
 Confirms correlation of roquin function with localization to P-bodies
Roquin interaction with P-body pathway independent of stress granules?

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

15. Looking for interaction partners: Co-Immunoprecipitation
Protein A/G binds antibody
Antibody binds antigen
Antigen binds to interaction partner

16. Roquin interacts with decapping proteins
Rck (essential for decapping)
Edc4 (decapping enhancer)
Dcp1a (decapping activator)
Co-Immunoprecipitation of roquin
Insensitive to RNase treatment
 mRNA independent

17. Roquin interacts with decapping proteins
Knockdown of Rck
ICOS expression increased
Model: roquin mediates silencing of ICOS through functional and physical interaction with proteins of the decapping pathway

18. Roquin-mediated repression does not require miRNA
Rck also essential in miRNA dependent post transcriptional signaling
 influence of miRNA on roquin-mediated ICOS downregulation to be checked

19. Roquin-mediated repression does not require miRNA & Dicer
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 rates
roquin induced similar decrease of ICOS mRNA expression in WT and Dicer-deficient cells

20. Roquin-mediated repression does not require miRNA & Dicer
 roquin down-regulation of ICOS mRNA is Dicer independent
ICOS and Hoxc8 mRNA expression measured
MEFs (WT & Dicer deficient)

21. Roquin-mediated repression does not require RISC
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

22. 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

23. Electrophoretic mobility shift assay

24. Binding position of roquin to ICOS mRNA
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

25. 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

26. Thank you for your attention!

27. Literature Presentation based on: Further literature:
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 Aug;11(8): 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 June ; 21(3): 403–408. doi: /j.ceb