Transcript RNA silencing

Posttranscriptional gene silencing
Transcriptional gene silencing (TGS)
Posttranscriptional gene silencing (PTGS)
• Promoters active
• Promoters silenced
• Genes hypermethylated in • Gene hypermethylated in
coding region
promoter region
•
Purpose
Viral
• Purpose - Viral
immunity?
immunity?
This has recently been termed “RNAi”
S. Grant Cell 96:303, 1999
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
• Has been used effectively in some animal models
S. Grant Cell 96:303, 1999
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
21-23 nt fragments
dsRNA
dsRNase
RNA degrading enzyme
RNA fragment
renders specificity
to the RNA
degrading enzyme
Carrington,Nature 408:150, 2000
RNA silencing• Model 2
RNA-directed RNA polymerases (RdRps) produces
“aberrant” RNA
2
Aberrant RNA produced
RdRp
viral ssRNA
1
RdRp
viral ssRNA
RdRp
plant mRNA
3
dsRNase
Carrington, Nature 408:150, 2000
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
Age of plant
Protected from
subsequent
infection
Infect with
blackring
nepovirus
No virus
1st infection
2ndinfection
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
NO2Nitrate reductase
35S promoter
nitrate reductase
= yellow
leaves
NH4
Transgene
Palauqui and Balzergue Curr. Biol. 9:59-66, 1999
RNA silencing
Observe:
35S promoter
nitrate reductase
or
nitrate reductase
35S promoter
or
nitrate reductase
Into plant leaves via
DNA-coated tungsten
Silencing of endogenous transgene
Conclusion:
Is DNA causing PTGS??
Palauqui and Balzergue Curr. Biol. 9:59-66, 1999
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
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