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Dies ist nur ein Beispielbild! Roquin binds inducible costimulator (ICOS) mRNA and effectors of mRNA decay to induce microRNA-independent post-transcriptional.

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Presentation on theme: "Dies ist nur ein Beispielbild! Roquin binds inducible costimulator (ICOS) mRNA and effectors of mRNA decay to induce microRNA-independent post-transcriptional."— Presentation transcript:

1 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 Aug;11(8): Kirstin Jurrat Paper Presentation, 1 st November 2010

2 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 mRNASummary & Discussion

3 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 (Rc3h1 san/san mice)

4 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

5 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

6 mRNA degradation

7 Degradation of eukaryotic mRNA: Decapping & P-bodies Seite 7 |

8 ICOS expression is controlled by roquin Seite 8 | In T H 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

9 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

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

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 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

13 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

14 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

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

16 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

17 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

18 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

19 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 rates roquin induced similar decrease of ICOS mRNA expression in WT and Dicer-deficient cells

20 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)

21 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

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 Seite 23 |

24 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

25 Summary & Discussion - in T H 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 |

26 Thank you for your attention!

27 Literature Presentation based on: Roquin binds inducible costimulator mRNA and effectors of mRNA decay to induce microRNA- independent post-transcriptional repression. Glasmacher EGlasmacher E, Hoefig KP, Vogel KU, Rath N, Du L, Wolf C, Kremmer E, Wang X, Heissmeyer V.Hoefig KPVogel KURath NDu LWolf CKremmer EWang XHeissmeyer 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 Seite 27 |


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