PostTranscriptional Gene Silencing PTGS

1
Post-Transcriptional Gene Silencing (PTGS)

• Also called RNA interference or RNAi
• Process results in down-regulation of a gene at
  the RNA level (i.e., after transcription)
• There is also gene silencing at the
  transcriptional level (TGS)
• Examples transposons, retroviral genes,
  heterochromatin

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Discovery of PTGS

• First discovered in plants (R. Jorgensen, 1990)
• Introduction of a transgene homologous to an
  endogenous gene often resulted in both genes
  being suppressed!
• Also called Co-suppression
• involved enhanced degradation of the endogenous
  and transgene mRNAs

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Discovery of PTGS (cont.)

• Involved attempts to manipulate pigment
  synthesis genes in petunia
• Genes were enzymes of the flavonoid/ anthocyanin
  pathway
• CHS chalcone synthase
• DFR dihydroflavonol reductase
• When these genes were introduced into petunia
  using a strong viral promoter, mRNA levels
  dropped and so did pigment levels in many
  transgenics.

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Flavonoid/anthocyanin pathway in plants
Strongly pigmented compounds
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DFR construct introduced into petunia CaMV - 35S
promoter from Cauliflower Mosaic Virus DFR cDNA
cDNA copy of the DFR mRNA (intronless DFR
gene) T Nos - 3 processing signal from the
Nopaline synthase gene
Flowers from 3 different transgenic petunia
plants carrying copies of the chimeric DFR gene
above. The flowers had low DFR mRNA levels in the
non-pigmented areas.
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Antisense Technology

• Antisense technology has been used for 20 years
• Based on introducing an antisense gene (or
  antisense RNA) into cells or organisms to try to
  block translation of the sense mRNA.
• Alternative to gene knock-outs, which are very
  difficult to do in higher plants and animals.

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• The antisense effect was probably due to RNAi
  rather than inhibiting translation.
• RNAi discovered in C. elegans (first animal)
  while attempting to use antisense RNA in vivo
• Control sense RNAs also produced suppression
  of target gene!
• sense (and antisense) RNAs were
  contaminated with dsRNA.
• dsRNA was the suppressing agent.

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Double-stranded RNA (dsRNA) induced interference
of the Mex-3 mRNA in the nematode C. elegans.
Antisense RNA (c) or dsRNA (d) for the mex-3
(mRNA) was injected into C. elegans ovaries, and
then mex-3 mRNA was detected in embryos by in
situ hybridization with a mex-3 probe. (a)
control embryo (b) control embryo hyb. with mex-3
probe
Conclusion dsRNA reduced mex-3 mRNA better than
antisense mRNA. Also, the suppression signal
moves from cell to cell.
Fig. 16.35
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PTGS occurs in wide variety of Eukaryotes

• called RNA interference or RNAi in
• C. elegans (nematode)
• Drosophila
• Mammalian cells
• called quelling in Neurospora
• not detected (yet) in Yeast!

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Mechanism of RNAi

• Some facts and findings
• Cells (plants and animals) undergoing RNAi
  contain small RNAs (25 nt) that seem to result
  from degradation of the target mRNA.
• A nuclease was purified from Drosophila embryos
  that digests target mRNAs
• the nuclease contained associated small RNAs
  (both sense and antisense)
• degradation of the small RNAs with micrococcal
  nuclease prevented the RNAi-nuclease from
  degrading target mRNA
• These facts suggest that a nuclease (Dicer)
  digests dsRNA into small fragments, which
  initiate the RNAi process by guiding the nuclease
  to the mRNA.

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Fig. 16.36
Generation of 21-23 nt fragments of target RNA in
a RNAi-competent Droso. embryo lysate/extract.
32P-labeled ds luciferase (luc) RNAs, either Pp
or Rr, were added to reactions 2-10 in the
presence or absence of the corresponding mRNA.
The dsRNAs were labeled on the sense (s),
antisense (a) or both (a/s) strands. Lanes 11, 12
contained 32P-labeled, capped, antisense Rr-luc
RNA.
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The dsRNA that is added dictates where the
destabilized mRNA is cleaved.
The dsRNAs A, B, or C were added to the
Drosophila extract together with a Rr-luc mRNA
that is 32P-labeled at the 5 end. The RNA was
then analyzed on a polyacrylamide gel and
autoradiographed.
Results the products of Rr-luc mRNA degradation
triggered by dsRNA B are 100nt longer than those
triggered by dsRNA C (and 100 nt longer again
for dsRNA A-induced degradation).
Fig. 16.37
13
High resolution gel analysis of the products of
Rr-luc mRNA degradation from the previous slide.
Results the cleavages occur mainly at 21-23 nt
intervals 14 of 16 cleavage sites were at a U.
There is an exceptional cleavage only 9 nt away
from the adjacent site (induced by dsRNA C) this
site had a stretch of 7 Us.
Conclusion Dicer cleaves at 23-nt intervals
after U.
Fig. 16.38
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Model for RNAi
By Dicer
21-23 nt RNAs
ATP-dependent Helicase?
Active siRNA complexes RISC
Very efficient process because many small
interfering RNAs (siRNAs) generated from a larger
dsRNA.
Fig. 16.39
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In plants, fungi, C. elegans Drosophila, a
RNA-dependent RNA polymerase (RDR) is involved in
initiation or amplification of silencing.
CBP and PABP block access for RDR.
PABP missing.
D. Baulcombe 2004 Nature 431356
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Why RNA silencing?

• Most widely held view is that RNAi evolved to
  protect the genome from viruses, and perhaps
  transposons or mobile DNAs.
• Some viruses have proteins that suppress
  silencing
• HCPro in plant potyviruses (first example)
• P19 in tomato bushy stunt virus binds to siRNAs,
  and prevents them from being recruited into the
  RISC.
• Tat protein in HIV

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Micro RNAs (MiRNAs)

• Recently, very small (micro) MiRNAs have been
  discovered in plants and animals.
• They resemble siRNAs, and they regulate specific
  mRNAs by promoting their degradation or
  repressing their translation.
• New use for the RNAi mechanism besides defense.

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Comparison of Mechanisms of MiRNA Biogenesis and
Action
DCL1 mutant
Better complementarity of MiRNAs and targets in
plants.
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Summary of differences between plant and
animal MiRNA systems Plants Animals of
miRNA genes 100-200 100-500 Location in
genome intergenic regions Intergenic regions,
introns Clusters of miRNAs Uncommon
Common MiRNA biosynthesis Dicer-like
Drosha, Dicer Mechanism of repression mRNA
cleavage Translational repression Location of
miRNA target in a gene Predominantly
Predominantly the 3'-UTR the open-reading
frame of miRNA binding sites in a target
gene Generally one Generally
multiple Functions of known target
genes Regulatory genes Regulatory
genescrucial crucial for development, for
development, structural enzymes proteins,
enzymes
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References

• Baulcombe, D. (2004) RNA silencing in plants.
  Nature 431 356-363.
• Millar, A.A. and P.M. Waterhouse (2005) Plant and
  animal microRNAs similarities and differences.
  Functional Integrative Genomics 5 129-135.