2. Advantages of C. elegans: 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction 4. transparent 5. only ~1000 cells 6. laser ablation 7. complete cell lineage map 8. genetics

6. RNA interference: A type of gene regulation Involving small RNA molecules and induced by double stranded RNA

7. Adding a Chalcone synthase gene from Petunia fused to a strong viral promoter to transgenic Petunia interfered with expression of the native homologous’ gene. Gene Silencing

8. 1.More common from strong promoters. 2. More common from inverted repeat inserts (could make a double stranded RNA). 3. Induced by RNA viruses (have double stranded RNA replication intermediates).

9. Tobacco plant expressing GFP protein Infected with RNA virus with GFP gene Virus infection travels through veins GFP expression inhibited starting at veins. RNA viruses can block expression of a transgene if a copy of the transgene has been added

10. Double stranded RNA is common to Viruses that can silence host gene expression And to transgenes that can silence host gene expression

11. Transgenes insert into chromosomal DNA randomly or

12. Transgenes often integrate in complexes

13. Gene silencing and RNA viruses share potential to produce dsRNA Fire and Mello used C. elegans to prove that dsRNA inhibits expression of endogenous genes homologous to that dsRNA

14. Double stranded GFP RNA interferes with expression of GFP in transgenic C. elegans GFP expressed in nuclei After adding dsRNA for GFP

15. Features of RNAi Induced by dsRNA with homology to exons Catalytic: very small amounts of ds RNA are sufficient Spreads: injection into gut silences genes in embryos Small RNAs produced

16. Mechanism of RNAi Dicer binds dsRNA And cleaves making siRNA siRNAs direct Risc to bind homologous mRNA and cleave it.

17. Catalysis: RdRP copies mRNA making more ds RNA. Dicer cuts that generating more siRNA More RdRP is activated and more dsRNA is made. Spread: dsRNA transported to other cells. Not in Drosophila or mammals

18. Double stranded GFP RNA interferes with expression of GFP in transgenic C. elegans GFP expressed in nuclei After adding dsRNA for GFP How would you design a genetic screen to identify the genes involved in the RNAi mechanism?

19. How would you set up a genetic screen to investigate the mechanism of RNAi? Design a screen for mutants in genes required for RNAi Easy to induce RNAi in C. elegans by feeding them E. coli engineered to express a transgene in both directions – sense and antisense

20. A real genetic screen for C. elegans genes essential for RNAi

21. A real genetic screen for C. elegans genes essential for RNAi

22. Candidate mutants isolated after feeding E. coli producing double stranded RNA How could double stranded RNA be made in E. coli? Checked candidate mutants by microinjection into gut The listed mutants passed the secondary test

24. Different mutants had Specific Phenotypes For activation of Transposable elements and silencing of transgenes

27. Functions of genes in RNAi decifered Rde4 = Initiating RNA binding protein Rde1 = Argonaut Rde2 = ? Mut7 = endonuclease

28. What is the function of RNAi mechanism in non-transgenic organisms? Protection against viruses Keep Transposable elements inactive Gene regulation

29. Gene regulation by small RNAs SiRNAs degrade mRNA to stop gene expression quickly StRNAs prevent translation to stop gene expression quickly

30. MIRNA gene HASTY AAA Pol II DCL1 HEN1, HYL1 AGO1 AAA.................. AAA DCL1 miRNA* RISC components RISC miRNA Target Cleaved target 57 unique miRNAs from 26 families ~200 MIRNA genes** **Cumulative data from Bartels, Carrington, Chen, Weigel, Zhu, others miRNA Precursor Transcript MIRNA genes are PolII genes that encode a hairpin shaped mRNA. Dicer cleaves the double stranded portion to make a short dsRNA. That combines with the RISC complex and directs cleavage of a specific target mRNA HYL1

31. miRNAs and Targets in Animals C. elegans114 Drosophila78 Zebrafish362 Mouse245 Human321 Registered miRNAs Fig. 3 from Lewis, Burge and Bartel (2005) Cell 120, 15-20 May target 1/3 of all genes Development - e.g. timing, stem cell function, differentiation Cell and organ identity Cancer - mis-regulation, deletion, duplication of MIRNA genes Herpesviruses SV40

32. Transcriptional gene silencing is initiated by RNA directed methylation of promoter regions dsRNA homologous to promoters leads to methylation and inactivation by recruitment of chromatin remodeling enzymes.

33. Centromeres are transcriptionally inactive. They have condensed chromatin Repeated sequences lead to production of double stranded RNAs The dsRNAs maintain the condensed state of the centromeres

34. Transcriptional Gene silencing requires dsRNA, DNA methyltransferases, histone methylation and histone deacetylation. RNA directed DNA methylation DNA methylases associate with histone modification enzymes leading to changes in histone methlyation patterns and histone deacetylation. Condensed chromatin results. Histone methylation

35. RNA is a gene regulator as well as a carrier of information

36. Transcriptional gene silencing is initiated by RNA directed methylation of promoter regions dsRNA homologous to promoters leads to methylation and inactivation by recruitment of chromatin remodeling enzymes.

37. Imprinting is another form of epigenetic gene regulation ICR – imprinting Control region Differential methylation leads to differential expression of Maternal and paternal alleles

38. Establishment of differential methylation CTCF zinc finger protein Protects ICR in female germline Denovo methylases Cannot methylate H19 Igf2 insulin-related growth factor Repressed in extraembryonic tissues and in some embryo tissues Imprinting

39. RNA directed methylases also affect methylation of genes in germline. May be important to imprinting

40. RNA can mediate differential expression of genes on same chromosome Mechanism not like RNAi because it works in cis. Like X inactivation Extraembryonic tissues: Igf2r and distant, linked genes Maternal ICR is methylated, Paternal alleles are not expressed. Paternal X chromosome Not expressed.

41. RNA directed regulation of gene expression is more common than we thought.