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Defensio Dissertationis Jörg Hackermüller

The RNA secondary structure dependence of RNA protein interactions and its implications for the post transcriptional regulation of gene expression. Defensio Dissertationis Jörg Hackermüller. AAAA. C. A A A A. A A A A. A A A A. C. C. C. degradation.

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Defensio Dissertationis Jörg Hackermüller

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  1. The RNA secondary structure dependence of RNA protein interactions and its implications for the post transcriptional regulation of gene expression Defensio Dissertationis Jörg Hackermüller

  2. AAAA C A A A A A A A A A A A A C C C degradation Regulation of Eukaryotic Gene Expression

  3. RNA-protein interactions • highly relevant (involved in many biological pathways – particularly in post transcriptional regulation of gene expression ) • often dependent on recognition of RNA (secondary) structure features • despite the importance no systematic investigation of RNA secondary structure dependence

  4. Quantitative model of RPIA • Model: • proteins binds only RNAs that form secondary structure feature • 1:1 binding (can be extended at cost of more complex expressions) • non pseudo-knotted RNA secondary structures (can be extended at cost of higher computational effort)

  5. NNUUNNUUU • A2 Ways to calculate p • NNUUNNUUU • HuR target mRNA • 5‘ • A1 • S(s) • S(A1,A2,s) • S(A1,s) • S(A2,s)

  6. A statistical test for the influence of secondary structure • (i) calculate p* [] for any test conformation  for all sequences in the set • (ii) calculate correlation coefficient between and p* [] • (iii) test whether this correlation is significant: • k number of sequences in set • r empirical correlation coefficient • t(.) Student’s t-distribution for (k-2) degs. of freedom • a significance level

  7. AAAAA mRNA stability regulation by HuR HuR HuR AAAAA AAAAA NPC neg. regulators (AUF1, TTP, …) • decay • Quiescent cells

  8. AAAAA AAAAA AAAAA AAAAA mRNA stability regulation by HuR HuR HuR AAAAA AAAAA NPC neg. regulators (AUF1, TTP, …) • decay • upregulation • Trigger • Quiescent cells

  9. HuR RNA recognition • HuR to RNA binding: • diverse affinities to target RNA (sub) sequences (Kd 120 pM to 13 nM) • Sequence properties: • HuR’s degenerate binding motif: N-N-U-U-N-N-U-U-U • binds in “all or nothing” manner (U9 1nM, U8 “nothing”1) 1< 0.01% of RNA bound

  10. HuR binds NNUUNNUUU in single stranded conformation • x unpaired • | paired • . No constraint ( opening base pair ) closing basepair

  11. modRNA mechanism • manipulate RNA-protein interactions through manipulation of RNA secondary structure • RNA secondary structure can change significantly upon hybridization of a second RNA (heteroduplex formation)

  12. modRNA mechnism II • RNA-RNA hybridization thermodynamically well understood • no model currently available for influence on RNA-protein interactions • RNA-RNA hybridization biologically very relevant for RNA-ligand interactions (non-protein coding RNAs) • approximate model of ternary equilibrium RNA – protein – short RNA • modRNA selected by calculating for any possible exactly reverse complementary modRNA of a given length (20nt)

  13. . . . . . . . . . . . . . . . . . . opener Op1 Openers specifically stabilize mRNA

  14. Specificity puzzle and modRNAs • Science

  15. Specificity puzzle and modRNAs • Fiction

  16. Outlook • endogenous modRNAs? • potential origin • bioinformatic strategies to find ‘em • modRNAs as tools in biology • may serve as complementary technique for RNAi • issue of delivery • modRNAs in drug discovery • modRNAs as pharmaceutic agents • modRNAs as tools in pharma research

  17. Summary People involved NIBR DT - IST Volker Uhl Jan-Marcus SeifertMartin Hintersteiner Nicole-Claudia Meisner Manfred Auer NIBR IK@N-ISS Torsten SchindlerSiegfried Höfinger Markus Jaritz Jörg Hackermüller Andras Aszodi Univ. Leipzig Peter F. Stadler Univ. Salzburg Fatima Ferreira Univ. Vienna Christoph Flamm Ivo Hofacker Kurt Gruenberger OeAW DOC 11/2000

  18. Backup slides Backup slides

  19. A quantitative model for RNA - ligand binding • Ligand binds only to RNA which has a particular accessible conformation RNA* • Experiments can not distinguish between accessible and non-accessible • Probability of accessible structures p* expressedby partition functions:

  20. modRNA model • neglect multiple binding of oligo O to mRNA M • O is exactly reverse complementary to M • no significant self complementarityin O and M • excess of O • Computation by constrained partition functions:

  21. modRNA model II • Effect of modRNAs on affinity

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