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From cyanide and water to RNA sensors of stress in human cytokine genes

From cyanide and water to RNA sensors of stress in human cytokine genes. Raymond Kaempfer, Hebrew University. Saladino et al. (2012). Cyanide and water will condense readily to formamide which can serve as the basis for all compounds needed to create RNA spontaneously.

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From cyanide and water to RNA sensors of stress in human cytokine genes

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  1. From cyanide and water to RNA sensors of stress in human cytokine genes Raymond Kaempfer, Hebrew University

  2. Saladino et al. (2012) Cyanide and water will condense readily to formamide which can serve as the basis for all compounds needed to create RNA spontaneously

  3. Water plus cyanide gives.. formamide

  4. Formamide: omnipotent precursor

  5. cGMP polymerizes in warm water to RNA as time goes by RNA goes on to evolve, as time goes by Abiotically formed cyclic precursors may actually have started their evolution toward complexity in a warm little pond, as first conceived by Darwin Ernesto Di Mauro and team, JBC, 2009

  6. Regulatory and template functions of RNA in modern organisms lend strong support to a scenario of kick-starting life throughRNA Kaempfer, Physics of Life Reviews, 2012

  7. RNA Sensors of Stress • Ability to respond to stress - critical • Integrated cellular stress response: heart of innate immunity • PKR: key stress kinase • PKR: focal point of host-virus interaction (viral anti-PKR strategies) • Novel principle: RNA activators of PKR • Discovery: in IFN-g and TNF-a genes

  8. Protective Th1 cell immunity IFN and TNF: Protective immunity Foreign invader / auto-antigen IL2 IFN TNF Work together: 5,000-fold synergy for immunity

  9. dsRNA binding domains N C dsRBM1 dsRBM1 dsRBM2 dsRBM2 dsRBM2 kinase kinase Spacer dimerization dimerization domains domain P P R Dimerization K K K K K K ATP ATP ATP ATP ATP R S Activation ATP RNA binding R R R R R P P R R R R R Activation of PKR requires dsRNA Inactive PKR Active PKR 33 bp needed for PKR activation transinter-phosphorylation

  10. PKR: a stress kinase • RNA-activated protein kinase (PKR) • Intracellular sensor of stress • Critical in control of cell growth, survival, host antiviral defense • Phosphorylates eIF2a • Inhibits translation by interfering with the ability of eIF2 to recycle between rounds of initiation

  11. Immune interferon

  12. Innate immune response G A A U P P C A C G U U G U A C U G C A P P PKR activated PKR 5’ ACUAGUCUUGUUAC a 3’ AUG AAAA eIF2 IFN-g P a inactivated eIF2 IFN-g / PKR Negative Feedback Loop IFN-g mRNA with pseudoknot in 5’-UTR Ben-Asouli et al., Cell, 2002

  13. Pseudoknot

  14. Restoration of the pseudoknot by compensatory mutations restores the ability to activate PKR a4 a4 / / - - RNA RNA ds ds wt wt a4r a4r a4 a4 a4r a4r PKR 68 68 - - eIF2a 38 38 - - Ben-Asouli et al., Cell, 2002

  15. 90 . . . C A A U U U A G U C A U C A C - G G A A A G A U C A G U A G U A U A 40 - U A C A 50 . C U - A K U G 100 - C G 80 U - A U - A U - A U U 60 30 U G C - G A A U . - A U U G C A - C G C - G C G U - A U G U U C A A U U 110 A Pseudoknot stem C U C A G U U - A C G - S1 20 U - A C U - A 70 C G - . G U G C - 5’ 120 ACUAGUC UUGUUAC A - U 10 170 180 U U C . U U U U G G G U C U U G G C G - A U U S2 A - U A C C C A G G A C C G C A U - C G U - G C S3 200 190 - A U 3’ - U A - G C G A U A 160 C C C A 130 . A U A U A U C G A U A U U A U A U G G C U G U U C U 150 140 U S3 helix: critical for PKR activation . Cohen-Chalamish et al., Nat Chem Biol 5:896-903, 2009

  16. Helix S3 is critical for PKR activation, including the A:U pair at the junction U U C U U . U U G G G U C U U G G C G U A C C C A G G A C C G C S3 U A U U U U 3’ A A A U U U G C G C G C U U U U U U C G C G C G G C G C G C A U G C G C G C A U A A A U U U . . . G G G U U U U U A C C C A G G A C C G U G C G C G C G U U U U A A A A C C C A G G A C C G U C A U UG C C C . . U U U 120 5’ AC U U U 3’ G G G C C C . . G G G C C C G G G C C C 200 A A A U U U 3’ 3’ 3’ U U U U U U RNA wt (ng/ml) -PKR -eIF2a orientation sensitive wt A202U U171A s3AU-J s3a - 0.1 0.25 0.1 0.25 0.1 0.25 0.1 0.25 0.1 0.25 The PKR activator structure in IFN-g mRNA extends deeply (78 nt) into the ORF

  17. The RNA sensor of PKR in human IFN-g mRNA • Our results imply a dynamic state in which the IFN-RNA sensor is disrupted by the passage of ribosomes and is regenerated readily • Efficient refolding of the RNA sensor, able to activate PKR, is facilitated by a combination of structural elements, including alternative conformations

  18. 90 . . . C A A U U U A G U C A U C A C - G G A A A G A U C A G U A G U A U A 40 - U A C A 50 . C U - A K U G 100 - C G 80 U - A U - A U - A U U 60 30 U G C - G A A U . - A U U G C A - C G C - G C G U - A U G U U C A A U U 110 A Pseudoknot stem C U C A G U U - A C G - S1 20 U - A C U - A 70 C G - . G U G C - 5’ 120 ACUAGUC UUGUUAC A - U 10 170 180 U U C . U U U U G G G U C U U G G C G - A U U S2 A - U A C C C A G G A C C G C A U - C G U - G C S3 200 190 - A U 3’ - U A - G C G A U A 160 C C C A 130 . A U A U A U C G A U A U U A U A U G G C U G U U C U 150 140 U S2 helix: critical for PKR activation . Cohen-Chalamish et al., Nat Chem Biol 5:896-903, 2009

  19. A U 3’ A U 121 C G A A 3’ U A 3’ A U 3’ U U 3’ G C A A U A A U U U A U C C G C C G G G A U G G C G G C C C G C A A U A A U U U U U U U U A A A U A G U G G C G G C C C C s2ab s2i PL S2 wt s2a s2b wt s2ab s2abAU-J 0 0 PKR- eIF2a- S2 helix: critical for PKR activation 3’ A U 3’ A U Asymmetric phenotype A U U A C G G C G C C G U U U A A U A A orientation sensitive => C C C G A:U junction pair is critical Anat Hasson

  20. Alternative RNA folding in the PKR activator: S2 helix 90 . . . C A A U U U A G U C A U C A C - G G A A A G A U C A G U A G U A U A 40 - U A C A 50 . C U - A K U G 100 - C G 80 U - A U - A U - A U U 60 30 U G C - G A A U . - A U U G C A - C G C - G C G U - A U G U U C A A U U 110 A Pseudoknot stem C U C A G U U - A C G - S1 20 U - A C U - A 70 C G - . G U G C - 5’ 120 ACUAGUC UUGUUAC A - U 10 170 180 U U C . U U U U G G G U C U U G G C G - A U U S2 A - U A C C C A G G A C C G C A U - C G U - G C S3 200 190 - A U 3’ - U A - G C G A U A 160 C C C A 130 . A U A U A U C G A U A U U A U A U G G C U G U U C U 150 140 U . Cohen-Chalamish et al., Nat Chem Biol 5:896-903, 2009

  21. Alternative RNA folding in the PKR activator UGAUCAG UGAUCAG 5’ UGAUCAG 5’ 3’ 3’ ACUAGUCUUGUUAC ACUAGUCUUGUUAC ACUAGUCUCAACCG . 120 120 120 5’ A U A U A U U A A C G A U G U G S3 C A A 3’ U wt A U A G A A U U C S3 U C G U G S3 C U G C U G A U A G U A 3’ C C G C as2a as2b U U G S3 C A U A A C aS2 is the dominant conformation aS2 S2 UGAUCAG 5’ ACUAGUCUCAACCG 120 A U A G U U G G C as2ab rPKR wt as2a as2b as2ab 0 RNA 68-

  22. K K aM3 90 90 M3 . . . U C A A A C U U U A G U C A . . . U C A A A C U U U A G U C A - - C G A A G A U C A G U A G U A C G G A A A G A U C A G U A G U A U A A U A 40 40 - K U A - 50 K C U A C A 50 A S1 . . C - U A C - U A 100 100 U G U G - - C G C G 80 80 - - U A U A - U A - U A P P S3 - - U A U U A U 60 30 60 U 30 U U - U - G C G C 5 5 5 ’ ’ ’ 5 ’ 3 ’ A A G G - A U - . A U . A A U U G U G U C C - A - G C A G C 3 ’ C C - - C G G C G G U U - - U A U A G G U U U U S1 aS2 A C A C A A A U U 110 110 U U PS A PS A C U C C U C A G A G U U S2 - S3 U A - U A - - C G C G S1 20 S1 20 - - U A U A C C - - U A 70 U A 70 - - C G C G . . 3’ 5’ G U G U 190 200 - - G C G C U 120 U A . 5’ C A C C C A G G A C C G - ACUAGUC UUGUUAC - A U ACUAGUC UUGUUAC A U 120 . 10 10 U - - - - - - 180 170 U U G G G U C U U G G C G A A C G A U G U C C U U U U aS2 U U U U U U G G G U C U U G G C G - . A U U 180 170 S2 A - A U U G - C G A C C A C C C A G G A C C G A U - G C 190 200 U U - A U A - U A C 3’ - G C G G A U 130 U C C C A . A A U A U A U C G A U C A 130 C C A U . A U A U A U C G A U U A A G G C U U U G U U C U A U 150 140 U U A A G G C U U U G U U 150 140 C U U Dynamic refolding of RNA . . G Cohen-Chalamish et al., Nature Chemical Biology, 2009

  23. PKR and the ribosome compete for mRNA Nature Chemical Biology

  24. 90 90 90 90 90 90 . . . . . . . . . . . . . . . A A A C C C U U U U U U U U U U U U U U U U U U A A A A A A G G G G G G U U U U U U C C C C C C A A A A A A U U U U U U C C C C C C A A A A A A A A A A A A A A A . . . C C C . . . A A A G G G A A A A A A A A A G G G A A A A A A U U U U U U C C C C C C A A A A A A G G G G G G U U U U U U A A A G G G A A A A A A . . . G G G U U U U U U 80 80 80 80 80 80 80 80 80 A A A A A A 40 40 40 40 40 40 A A A 50 50 50 50 50 50 A A A A A A K K K K K K G G G G G G G G G G G G C C C C C C G G G G G G A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A C C C C C C C C C C C C G G G G G G G G G A A A A A A A A A A A A U U U U U U A A A A A A C C C C C C C C C C C C U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U G G G G G G C C C C C C C C C U U U U U U A A A A A A U U U U U U U U U U U U G G G G G G G G G G G G C C C C C C C C C A A A A A A C C C C C C C C C C C C U U U U U U A A A A A A A A A U U U U U U G G G G G G A A A A A A A A A A A A A A A A A A C C C C C C C C C C C C C C C G G G G G G 100 100 100 100 100 100 70 70 70 70 70 70 G G G G G G G G G U U U U U U U U U G G G G G G G G G U U U U U U A A A A A A 60 60 60 60 60 60 U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U 30 30 30 30 30 30 Pseudoknot Pseudoknot Pseudoknot Pseudoknot Pseudoknot Pseudoknot A A A A A A U U U U U U . . . . . . Stem Stem Stem Stem Stem Stem U U U U U U G G G G G G C C C C C C G G G G G G 3 3 3 3 3 3 ’ ’ ’ ’ ’ ’ G G G G G G C C C C C C 5 5 5 5 5 5 ’ ’ ’ ’ ’ ’ 20 20 20 20 20 20 200 200 200 200 200 200 190 190 190 190 190 190 U U U U U U A A A A A A C C C C C C U U U U U U U U U U U U U U U U U U A A A A A A U U U U U U AGA AGA AGA AGA AGA AGA AGUCU AGUCU AGUCU AGUCU AGUCU AGUCU ACUAGUC ACUAGUC ACUAGUC ACUAGUC ACUAGUC ACUAGUC UUGUUAC UUGUUAC UUGUUAC UUGUUAC UUGUUAC UUGUUAC A A A A A A C C C C C C C C C C C C C C C C C C A A A A A A G G G G G G G G G G G G A A A A A A C C C C C C C C C C C C G G G G G G C C C C C C . . . . . . . . . . . . . . . . . . U U U U U U 10 10 10 10 10 10 A A A A A A UCU UCU UCU UCU UCU UCU UCGGA UCGGA UCGGA UCGGA UCGGA UCGGA AACGAUG AACGAUG AACGAUG AACGAUG AACGAUG AACGAUG U U U U U U G G G G G G G G G G G G G G G G G G U U U U U U C C C C C C U U U U U U U U U U U U G G G G G G G G G G G G C C C C C C G G G G G G U U U U U U U U U U U U C C C C C C U U U U U U C C C C C C A A A A A A U U U U U U U U U U U U 170 170 170 170 170 170 A A A A A A U U U U U U S3 S3 S3 S3 S3 S3 S1 S1 S1 S1 S1 S1 S2 S2 S2 S2 S2 S2 120 120 120 120 120 120 180 180 180 180 180 180 A A A A A A U U U U U U 110 110 110 130 130 130 130 130 130 A A A A A A U U U U U U G G G G G G C C C C C C U U U U U U . . . . . . A A A A A A C C C C C C U U U U U U G G G G G G A A A A A A U U U U U U 140 140 140 140 140 140 160 160 160 160 160 160 C C C C C C U U U U U U C C C C C C U U U U U U U U U U U U G G G G G G A A A A A A A A A A A A C C C C C C A A A A A A C C C C C C G G G G G G A A A A A A U U U U U U U U U U U U A A A A A A U U U U U U A A A A A A C C C C C C U U U U U U U U U U U U G G G G G G G G G G G G C C C C C C U U U U U U U U U U U U U U U U U U U U U U U U 150 150 150 150 150 150 IFN- RNA activator of PKR <= 33 bp 33 bp needed for PKR activation

  25. Tumor necrosis factor

  26. ex 1 ex ex 3 A n Inflammatory response, apoptosis 2-APRE Splicing control element (104 nt) Splicing TNF-a pre-mRNA TNF-a mRNA TNF-a Activation of PKR PKR gene induced PKR activated PKR inactive Human TNF-a gene Positive feedback loop 5 ’ 2 ex 4 3’UTR ARE Osman et al., Genes Dev, 1999

  27. Regulatory and template functions of RNA in modern organisms lend strong support to a scenario of kick-starting life throughRNA

  28. Smadar Cohen-Chalamish Lise Sarah Namer Farhat Osman Yitzhak Ben-Asouli Yona Banai Anat Hasson Dahlia Weinberg kaempfer@hebrew.edu Faculty of Medicine The Hebrew University of Jerusalem

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