1. Regulatory Non-Coding RNAs Yair Aaronson 22.12.11

2. Types of RNAs mRNA tRNA rRNA snoRNA PiRNA miRMA siRNA

3. MicroRNAs ~1300 miRs in human "One to many” Known Biogenesis and processing mechanism Essential (e.g. Embryogenesis)

4. Chromosome X Inactivation

5. HOX Transcript Antisense Intergenic RNA (HOTAIR)

6. Models of Gene Regulation by ncRNAs Trans-actingCis-acting

7. Scheme for the PRC (Polycomb repressive Complexes) signaling. Choi et al. 2010.

8. Histon Modifications is

9. RNA folding Prediction Vienna RNAfold Algorithm Michael Zuker and Patrick Stiegler. 1981

10. Maintenance of pluripotencyLinage commitment Differentiation and development Maintain cell identityNeurological diseasesGenes SilencingX chromosome inactivationCancer Regulatory ncRNAs Epigenetic modifications Transcription control Post-transcriptional processing Nuclear trafficking

11. Detection of Short RNAs in Polycomp Repressed Targets Kanhere et al. 2010

12. Short RNAs were concentrated at the 5’ end of genes

13. PRC2 – short-RNAs Interactions

14. Electrophoretic Mobility Shift Assay (EMSA)

15. In Vitro Interactions of Short RNAs and Chromatin Remodelers (PRC2)

16. PRC2 interacts with short, promoter-associated RNAs But these interactions don’t necessarily leads to detectable H3K27me3

17. Validation – PRC2 binding short RNAs case gene repression in cis

18. New class of non-coding short RNAs found in Polycomb targets, causing genes repression in cis by stabilizing the association of RPC2 with chromatin. The model does not explain why some of the smRNAs containing genes, that interact with PRC2, remain free of H3K27me.

19. RNAs containing different sequences may interact with similar RNA-binding proteins Finding similar RNAs function by searching common secondary structure. Bivalent genes – epigenetic transcription activation (H3K4me3) and repression (H3K27me3) at the same gene – allowing regulatory ncRNAs to be transcribed. ncRNAs can function in a similar way to protein - mediators and TFs.

20. Long Intergenic Non-Coding (linc) RNA ~1600 bases large, multi-exonic RNAs.

21. Intergenic K4–K36 domains produce multi-exonic RNAs Guttman et al. 2009

22. Codon Substitution Frequencies (CSF) Score

23. lincRNAs K4-K36 domains do not encode proteins and are conserved Protein-coding - exons Protein coding - introns lincRNAs - exons Known ncRNAs

24. Checking for lincRNAs function For each lincRNA, 5 different shRNA lenti viral vectors were designed and tested (1143 hairpins). The one with the best repressive effect was chosen. Gene expression microarray for each lincRNA knockdown. Knockdowns for: 147 lincRNA. PC: 40 known ES proteins. NC: 27 different shRNAs with no known cellular target.

25. lincRNAs affect global gene expression proteinlincRNA 95%93% of knockdown caused significant impact on the expression of >10 genes 207175average of protein- coding transcripts affected

26. lincRNAs are critical for the maintenance of pluripotency. Identification of 26 lincRNAs that had major effects on endogenous Nanog levels, using Nonog- Luciferase construct. Guttman et al. 2011

27. lincRNAs seem to affect expression largely in trans (as TFs). (trans acting) lincRNAs can either repress or activate genes

28. lincRNAs repress specific differentiation lineages. Identification of 30 lincRNA that serves as differentiation barriers. >85% of the lincRNAs associated with specific differentiation lineages showed upregulation of the well-known marker genes for the identified states. Comparing the expression profile of the KD cells vs knows lineages expression profile.

29. lincRNAs Physically Interact With Chromatin Regulatory Proteins. Examine genes expression after KD each of the lincRNAs – protein pairs – showed highly significant overlap in affected gene expression in >40%

30. “flexible scaffold” A model for lincRNA integration into the molecular circuitry of the cell

31. Using chromatin signature, ~3800 lincRNAs were identified. 226 embryonic stem-cells associated lincRNAs. lincRNAs affect global gene expression.

32. Maintenance of pluripotencyLinage commitment Differentiation and development Maintain cell identityNeurological diseasesGenes SilencingX chromosome inactivationCancer Regulatory ncRNAs Epigenetic modifications Transcription control Post-transcriptional processing Nuclear trafficking

33. ncRNAs can compete on miRNAs (Sponges) Margaret S. Ebert and Phillip A. Sharp. 2010

34. Marcella et al. 2011

35. ceRNA Hypothesis – RNA Transcripts Communication Salmena et al. 2011

36. lncRNAs and enhancers Ulf Andersson Ørom, Ramin Shiekhattar. 2011

37. Mercer et al. 2009

38. summary ncRNA are being recognized as one of the important functional players of the genome, rather than the ‘junk’ RNA approach. Approximates say that there are many tens of thousands of regulatory ncRNAs. Obtaining a diverse set of mechanisms, ncRNAs may act as regulators of almost all biological aspects. New gene definition. How is a small repertoire of chromatin remodeling complexes (with little DNA sequence specificity) able to specify the complex array of chromatin modifications?

39. What's next? Who is the major gene expression regulator? (Wilson, M. D. et al. 2008) The role of ncRNAs in diseases? Functional prediction? Evolution of ncRNA. Next Generation Sequencing, live cell RNA imaging, RNA proteomics and RNA structural biology techniques, will allow a better understanding in the near future!

40. Thanks