Posttranscriptional gene silencing

• Promoters silenced• Genes hypermethylated in

promoter region • Purpose - Viral

immunity?

• Promoters active• Gene hypermethylated in

coding region• Purpose - Viral

immunity?

S. Grant Cell 96:303, 1999

Transcriptional gene silencing (TGS) Posttranscriptional gene silencing (PTGS)

This has recently been termed “RNAi”

Posttranscriptional gene silencing• 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

S. Grant Cell 96:303, 1999

• Has been used effectively in some animal models

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

RNA silencing-

• Model 1

dsRNase

dsRNA

21-23 nt fragments

RNA fragment renders specificity to the RNA degrading enzyme

RNA degrading enzyme

Carrington,Nature 408:150, 2000

RNA silencing-

• Model 2

viral ssRNA

Carrington, Nature 408:150, 2000

RNA-directed RNA polymerases (RdRps) produces “aberrant” RNA

RdRp

dsRNase

3

1

Aberrant RNA produced

viral ssRNA

plant mRNA

2

RdRp

RdRp

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?

Recovery from plant infection and immunity

No virus

Infect with blackring nepovirus

1st infection 2ndinfection

Protected from subsequent infection

Age of plant

Ratcliff, Science 276:1558, 1997

Plant immunity likely spread through plasmodesmata

• Plasmodesmata directly connect cytoplasm of adjacent cells

– Animal cells use gap junctions for communication

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

RNA silencing- viruses fight back• Viral protein p25is a movement protein that prevents cellular

RdRp activity

RNA silencing

• Viruses come in DNA or RNA forms

35S promoter nitrate reductase

Nitrate reductase

NO2-

NH4

= yellow leaves

Transgene

Palauqui and Balzergue Curr. Biol. 9:59-66, 1999

RNA silencing

Palauqui and Balzergue Curr. Biol. 9:59-66, 1999

Silencing of endogenous transgene

Into plant leaves via DNA-coated tungsten

35S promoter nitrate reductase

35S promoter

nitrate reductase

or

nitrate reductaseor

Conclusion:Is DNA causing PTGS??

Observe:

RNA silencing by ectopic pairingThree phases suggested:

Introduced DNA pairs with homologous gene

Transcriptional interference- aberrant RNA produced

Aberrant RNA spreads to adjacent cells through plasmodesmata

Alternative models for PTGS

RNA silencing

• 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