Das zentrale Dogma der Molekularbiologie

Transkript

1 Das zentrale Dogma der Molekularbiologie DNA makes RNA makes protein Francis Crick, 1956

2 Der komplexe Lebenszyklus einer RNA

3 Der Aufbau einer eukaryotischen mrna transkribierte Region DNA RNA translatierte Region

4 Der Aufbau einer eukaryotischen mrna transkribierte Region DNA gespleißte RNA

5 Alternatives Spleißen (AS) erzeugt mrna- und Protein-Isoformen Transkription Spleißen Translation Genom pre-mrna mrna Protein Abbildung aus &

6 Beispiel I: Proteindiversifizierung von Dscam in Drosophila neuronaler Zelloberflächen-Rezeptor mehr als verschiedene Isoformen Interaktion identischer Rezeptoren vermittelt Abstoßung Zipursky et al. (2006) Trends Biochem Sci 10:581-8; Hemani and Soller (2012) Biochem Soc Trans 40: 804-9

7 Beispiel II: AS als Schalter in embryonalen Stammzellen Erhalt der Pluripotenz Differenzierung Gabut et al. (2011) Cell 147,

8 RNA-bindende Proteine (RBPs) bestimmen die Spleißreaktion Das Spliceosom katalysiert das Entfernen der Introns Keren et al., Nat Rev Genet, 2010

9 RNA-bindende Proteine (RBPs) bestimmen die Spleißreaktion Das Spliceosom katalysiert das Entfernen der Introns Keren et al., Nat Rev Genet, 2010

10 Genomic Views of Splicing Regulation Image: Heng-Chang Chen Institute of Molecular Biology (IMB), Mainz

11 Gene expression is extensively controlled at the posttranscriptional level

12 Alternative splicing is crucial to generate proteome diversity Important for tissue identity & targeted in genetic disorders and cancer

13 cis-regulatory elements encode splicing regulation Spliceosome (U2 subunit)

14 RNA-binding proteins promote or repress spliceosome recruitment How the cis-elements are interpreted by RNA-binding proteins is called the splicing code

15 Long-term aims of my research Decipher the regulatory code of splicing. Predict, understand and treat erroneous splicing in genetic disease and cancer.

16 3 splice site definition Ribosome-associated quality control Splicing cis-regulatory elements and the splicing code Translation Alu element exonization in evolution and disease

17 Project 1: Ribonucleoprotein complex assembly 3 splice site definition Splicing Translation How do ribonucleoprotein complexes assemble at the 3 splice site?

18 U2AF65 is a central player in 3 splice site definition Spliceosome (U2 subunit) How does U2AF65 specifically recognize splice sites?

19 in vitro iclip reveals the intrinsic binding activity of U2AF65 iclip König et al., NSMB 2010 Sutandy et al., Genome Res 2018

20 In vitro vs. in vivo comparison pinpoints regulatory hotspots What causes the differences?

21 Machine learning predicts regulators of U2AF65 binding Predicted regulators Binding at cleared & stabilized sites

22 PTBP1 and FUBP1 shape U2AF65 binding

23 in vitro iclip uncovers regulation of U2AF65 by co-factors Sutandy et al., Genome Res 2018

24 Project 2: The regulatory code of splicing Decipher the regulatory network of selected splicing events Predict disease-causing mutations Splicing cis-regulatory elements and the splicing code Translation

25 Aberrant splicing of RON promotes invasive cancer cells We aim for a complete map of cis-regulatory elements that control RON splicing

26 High-throughput screen for cis-regulatory elements

27 Quantification of mutation effects with nucleotide resolution Py-tract 3 splice site cis-regulatory element (hnrnph)

28 The dense regulatory landscape of RON exon 11 Exon 10 Exon 11 Exon 12

29 The identified mutations modulate splicing in cancer patients COSMIC (Catalogue of Somatic Mutations in Cancer) TCGA (The Cancer Genome Atlas)

30 hnrnp H has several binding sites around RON exon 11

31 Epistatic interactions link RBPs to cis-regulatory elements Exon 11 inclusion (%) upon HNRNPH knockdown Exon 11 inclusion (%) in control cells

32 Epistatic interactions link RBPs to cis-regulatory elements Exon 11 inclusion (%) upon HNRNPH knockdown Stronger reaction Weaker reaction to knockdown Exon 11 inclusion (%) in control cells

33 Mutants in hnrnp H binding sites are less affected by knockdown Exon 11 inclusion (%) upon HNRNPH knockdown Weaker reaction to knockdown Exon 11 inclusion (%) in control cells

34 Epistatic effects point to most relevant hnrnp H binding sites Cooperative binding?

35 hnrnp H shows cooperative binding in the alternative exon

36 hnrnp H levels drive a switch-like splicing response

37 Cooperative binding results in a switch-like splicing response Cooperative binding Switch-like splicing Braun et al., Nat Commun, under revision

38 Thank you! König group: Heike Hänel Simon Braun FX Reymond Sutandy Andrea Hildebrandt Stefanie Ebersberger Mariela Cortes Lopez Laura Schulz Andreas Buchbender Holger Mutter Kathi Zarnack (BMLS Frankfurt) Samarth Thonta Setty Michael Sattler (TU Munich) Hyun Seo Kang Stefan Legewie (IMB) Mihaela Enculescu Lu Huang Petra Beli (IMB) Nuno Barbosa Morais (Lisbon) Bernardo de Almeida IMB administration IMB core facilities Maria Mendez-Lago (Genomics) Anke Busch (Bioinformatics)

39 Thank you! 3 splice site definition Ribosome-associated quality control Splicing cis-regulatory elements and the splicing code Translation Alu element exonization in evolution and disease