Carl T. Bergstrom Department of Biology University of Washington Seattle, WA

1. Can gene regulation be strategically robust against internal subversion? Insights from RNAi and other immune systems Carl T. Bergstrom Department of Biology University of Washington Seattle, WA Workshop on the evolutionof gene regulatory logic Santa Fe InstituteJanuary 6-8, 2006

2. Acknowledgements Rustom AntiaEmory University Tom Daniel Ben Kerr David Krakauer Michael Lachmann Erin McKittrick Jevin West

3. Introduction: The immunity problem

4. A teleological approach to immune function • The job or function of the immune system is to: • Clear pathogens.

5. A teleological approach to immune function • The job or function of the immune system is to: • Reduce the damage that pathogens cause(Matzinger)

6. A teleological approach to immune function • The job or function of the immune system is to: • Reduce the damage that pathogens cause(Matzinger) • …without using undue energy or resources. (Schmid-Hempel)

7. To function effectively, an immune system must feature: • Learning: What is self and what is non-self? • Sensitivity: Respond early to infection • Breadth: Recognize any new pathogen • Specificity: Generate highly specific responses. • Amplification: Generate a large-scale response • Coordination: Regulate a large-scale response • Memory: Make good use of prior experience. • This is already a very hard control problem…

8. Efficiency and the immune response • How does the immune system optimally allocate its effort and manage the balance between conserving resources and reducing damage from pathogens? (Schmid-Hempel, Segel, etc.)

9. Two additional challenges Avoid autoimmunity: Immune systems cannot afford false positives! • Avoid subversion Pathogens have evolved to disrupt immune surveillance and control by subterfuge, sabotage, subversion.

10. Example: chemokine signalling • E.g. many vertebrate viruses - especially pox viruses - interfere with the chemokine signals that the immune system uses for regulation and control. • Produce proteins to stimulate chemokine receptors • Alter the host’s chemokine expression • Produce decoy chemokine receptors • Block chemokine receptors • Sabotage chemokine signals (Liston and McColl 2003)

11. Example: Viroid pathogenesis Plant viroids, small 200-400 nt non-coding RNA virus-like pathogens, do not code for genes but they do cause symptoms. How? Viroids encode RNAs that stimulate the RNAi system to turn against the host and knock out the expression of crucial host genes. (Wang et al 2004 PNAS)

12. Constraints on immune function: Avoid autoimmunity Avoid subversion Opposition Goal: reduce damage from pathogens

13. Robustness and strategic robustness • Robustness: Preserve phenotype or function despite broad variation in environmental conditions, despite noise, and despite internal component failure. • Strategic robustness: Preserve phenotype or function despite internal attempts to sabotage the system.

14. A comparative framework • Vertebrate adaptive immunity • Vertebrate and invertebrate innate immunity • Plant immunity • Social insect colony recognition. • RNA interference (RNAi) • Restriction modification ...and still to be discovered?

15. RNA interference

16. How do immune systems generate pathogen-specific responses? Immune cell Pathogen

17. Adaptive fit hypothesis antibody B-cell

18. clonal expansion Clonal selection hypothesis B-cell clone 1 clone 2 clone 3 clone n ( repertoire n very large)

19. Building a plasticine template is very hard with protein PROTSCAPE, University of Bath

20. Templating comes naturallywith nucleic acids

21. How viruses replicate

22. Dicer dsRNA is detected... ...and degraded. siRNA In a virus-infected cell dsRNA

23. In a virus-infected cell Half of the fragments are complementary to viral mRNAs. siRNA mRNA

24. In a virus-infected cell Half of the fragments are complementary to viral mRNAs. They are stabilized by RISC proteins. siRNA + RISC

25. In a virus-infected cell Half of the fragments are complementary to viral mRNAs. They are stabilized by RISC proteins. And they bind to the viral mRNAs.

26. Argonaute The viral mRNAs are then destroyed by Argonaute,knocking out viral protein expression. In a virus-infected cell

27. RDRP Alternatively... A host enzyme RDRP copies the viral mRNA new dsRNA Dicer And then Dicer takes over. Amplification! In a virus-infected cell

28. Parallel to the vertebrate immune system • Discriminate non-self from self • Generate a highly specific response against non-self elements. • Massively amplify the response to meet the threat.How does RNAi avoid autoimmunity and yet deter pathogen subversion?

29. A simple ODE model

30. dsRNA mRNA+RISC complex RISC mRNA RNAi as a molecular control system Viral infection or accidental generation Degradation Transcription

31. dsRNA degrade complex RISC mRNA mRNA A system of ODEs State variables: RNA species

32. dsRNA degrade complex RISC mRNA mRNA Two equilibria: 1) No silencing 2) Silenced R0 determines stability.

33. dsRNA degrade complex RISC mRNA mRNA Starting with high initial dsRNA concetration

34. dsRNA degrade complex RISC mRNA mRNA Low initial dsRNA concentration

35. Unidirectional amplification

36. RDRP Only upstream elements are actually copied. orignal primer new dsRNA RDRP copies RNA in a unidirectional manner

38. Multitype branching process model Track contiguous siRNAs: A, B, C, D ,E , F, G, ... d: siRNA destroyed p: nothing happens q: RDRP falls off Leading eigenvalue is (1- d) Corresponding eigenvector is (1,0,0,0,...,0) Right eigenvector is (0,0,0,...,0,1) Mean behavior (1- d) 0 0 ... (1- d)(1- p) (1- d) 0 ... (1- d)(1- p)(1- q) (1- d)(1- p) (1- d) ... ... ... ... ...

39. Mean trajectory of the system 5 segments. Start with one "E". Parameters: d=0.2, p=0. q=0

40. dsRNA mRNA+RISC complex RISC mRNA A polar model of RNA silencing Viral infection or accidental generation Degradation Transcription

41. dsRNA degrade complex RISC mRNA mRNA A system of 3n+1 ODEs. Track n distinct RNA segments

42. dsRNA degrade complex RISC mRNA mRNA A small initial dose

43. dsRNA degrade complex RISC mRNA mRNA A higher initial dose

44. dsRNA degrade complex RISC mRNA mRNA Continual mRNA production

45. Immune system as amplifier

46. Multiple safeguards

47. ADAR Dicer ADARs and localization Virus Nucleus Cytoplasm Reduces the probability that a reaction is initiated, without changing the course of the reaction if does begin.

48. ERI-1 induces a threshold Imposes a threshold for initiating a reaction

49. dsRNA degr. complex RISC mRNA mRNA Systemic silencing Ring vaccination at the cellular level

50. dsRNA dsRNA degr. degr. complex complex RISC mRNA mRNA RISC mRNA mRNA dsRNA dsRNA degr. degr. complex complex RISC mRNA mRNA RISC mRNA mRNA dsRNA dsRNA degr. degr. complex complex RISC mRNA mRNA RISC mRNA mRNA Systemic silencing Systemic spread of the silencing signal.