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Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction

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. RNA interference: A type of gene regulation Involving small RNA molecules

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Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction

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  1. 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

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

  3. 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

  4. Gene Silencing • 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).

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

  6. 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

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

  8. 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

  9. Mechanism of RNAi Dicer binds dsRNA And cleaves making siRNA siRNAs direct Risc to copy homologous mRNA Dicer binds short stretch of dsRNA and cleaves it.

  10. 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

  11. 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?

  12. The real genetic screen for C. elegans genes essential for RNAi

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

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

  15. 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 MIRNA gene Pol II AAA DCL1 HYL1 ~200 MIRNA genes** DCL1 HEN1, HYL1 57 unique miRNAs from 26 families HASTY AGO1 RISC components miRNA Precursor Transcript miRNA* AGO1 RISCmiRNA Target AAA ..... ............. AGO1 **Cumulative data from Bartels, Carrington, Chen, Weigel, Zhu, others Cleaved target AAA

  16. 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 miRNAs and Targets in Animals Registered miRNAs C. elegans 114 Drosophila 78 Zebrafish 362 Mouse 245 Human 321 Herpesviruses SV40

  17. 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.

  18. Expression of double stranded RNA leads to chromatin remodeling

  19. 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

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

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