Posttranscriptional gene silencing

1. Promoters silenced Genes hypermethylated in promoter region Purpose - Viral immunity? Promoters active Gene hypermethylated in coding region Purpose - Viral immunity? Posttranscriptional gene silencing Transcriptional gene silencing (TGS) Posttranscriptional gene silencing (PTGS) This has recently been termed “RNAi” S. Grant Cell 96:303, 1999

2. Degrades all RNA transcripts that are homologous, including unlinked genes Observed in filamentous fungi, ciliates, and animals Injection of dsRNA into worms or fruit fly inhibits endogenous gene expression Posttranscriptional gene silencing • Has been used effectively in some animal models S. Grant Cell 96:303, 1999

3. RNA silencing- • plant “immunity” to viral infection involves spread of signals across membranes • silencing triggered by dsRNA, transgenes, viruses • If virus carries a plant gene, that plant gene is also silenced • “Recovery”- when virus degrades the virus entirley, and is now protected against infection by that virus

4. 21-23 nt fragments dsRNA RNA degrading enzyme dsRNase RNA fragment renders specificity to the RNA degrading enzyme RNA silencing- • Model 1 Carrington,Nature 408:150, 2000

5. Aberrant RNA produced RdRp viral ssRNA viral ssRNA 3 2 1 RdRp plant mRNA dsRNase RNA silencing- RNA-directed RNA polymerases (RdRps) produces “aberrant” RNA • Model 2 RdRp Carrington, Nature 408:150, 2000

6. Problem with models - • Promoterless DNA can induce PTGS • Not all genes undergo PTGS • How can PTGS spread through an entire plant?? • What does an “aberrant” RNA look like?

7. Protected from subsequent infection 1st infection 2ndinfection Recovery from plant infection and immunity Age of plant Infect with blackring nepovirus No virus Ratcliff, Science 276:1558, 1997

8. Plant immunity likely spread through plasmodesmata • Plasmodesmata directly connect cytoplasm of adjacent cells • Animal cells use gap junctions for communication

9. RNA silencing- viruses fight back • Virus spreads rapidly through plant via opening channels in the cell wall • e.g. Potato virus X (an RNA virus) has three movement proteins- p25, p12 and p8 • Viral protein p25 is a movement protein that also prevents cellular RdRp activity • Thus, virus spreads by two proposed mechanisms: • opening channels to adjacent to cells • inhibiting RNA silencing • Note- viral RdRp does not result in silencing

10. RNA silencing- viruses fight back • Viral protein p25is a movement protein that prevents cellular RdRp activity

11. NO2- Nitrate reductase = yellow leaves NH4 RNA silencing • Viruses come in DNA or RNA forms 35S promoter nitrate reductase Transgene Palauqui and Balzergue Curr. Biol. 9:59-66, 1999

12. 35S promoter nitrate reductase or 35S promoter nitrate reductase or nitrate reductase Into plant leaves via DNA-coated tungsten RNA silencing Observe: Silencing of endogenous transgene • Conclusion: • Is DNA causing PTGS?? Palauqui and Balzergue Curr. Biol. 9:59-66, 1999

13. RNA silencing by ectopic pairing Three phases suggested: Introduced DNA pairs with homologous gene Transcriptional interference- aberrant RNA produced Aberrant RNA spreads to adjacent cells through plasmodesmata

14. RNA silencing Alternative models for PTGS • DNA pairing activates RNA degradation • dsDNA “looks like “ aberrant dsRNA and so activates RNA degradation • hypermethylation leaves “mark” on transgene for formation of aberrant transcripts