Post-Transcriptional Gene Silencing (PTGS)

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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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• PTGS is heritable, although it can be modified in
  subsequent cell divisions or generations
• Ergo, it is an epigenetic phenomenon

Epigenetics - refers to heritable changes in
phenotype or gene expression caused by mechanisms
other than changes in the underlying DNA sequence.
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Antisense Technology

• Used from 1980 on, to repress specific genes
• Alternative to gene knock-outs, which were/are
  very difficult to do in higher plants and animals
• Theory by introducing an antisense gene (or
  asRNA) into cells, the asRNA would zip up the
  complementary mRNA into a dsRNA that would not
  be translated
• The antisense effect was highly variable, and
  in light of the discovery of RNAi, asRNA
  probably inhibited its target by inducing RNAi
  rather than inhibiting translation.

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Discovery of PTGS

• First discovered in plants
• (R. Jorgensen, 1990)
• When Jorgensen introduced a re-engineered gene
  into petunia that had a lot of homology with an
  endogenous petunia gene, both genes became
  suppressed!
• Also called Co-suppression
• Suppression was mostly due to increased
  degradation of the mRNAs (from the endogenous and
  introduced genes)

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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, but gene was still being
transcribed.
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• RNAi discovered in C. elegans (first animal)
  while attempting to use antisense RNA in vivo
• Craig Mello Andrew Fire (2006 Nobel
  Prize in Physiology Medicine)
• Control sense RNAs also produced suppression
  of target gene!
• sense 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
Conclusions (1) dsRNA reduced mex-3 mRNA better
than antisense mRNA. (2) the suppressing signal
moved from cell to cell.
Fig. 16.29
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PTGS (RNAi) occurs in wide variety of Eukaryotes

• Angiosperms
• C. elegans (nematode)
• Drosophila
• Mammalian cells
• Chlamydomonas (unicellular
• Neurospora, but not in Yeast!

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Mechanism of RNAi Role of Dicer

• Cells (plants and animals) undergoing RNAi
  contained small fragments (25 nt) of the RNA
  being suppressed.
• A nuclease (Dicer) was purified from Drosophila
  embryos that still had small RNA fragments
  associated with it, both sense and antisense.
• The Dicer gene is found in all organisms that
  exhibit RNAi, and mutating it inhibits the RNAi
  effect.
• Conclusion Dicer is the endonuclease that
  degrades dsRNA into 21-24 nt fragments, and in
  higher eukaryotes also pulls the strands apart
  via intrinsic helicase activity.

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Fig. 16.30
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. 16.31
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Model for RNAi
By Dicer
21-23 nt RNAs
ATP-dependent Helicase or Dicer
Active siRNA complexes RISC - contain
Argonaute instead of Dicer
Very efficient process because many small
interfering RNAs (siRNAs) generated from a larger
dsRNA.
Fig. 16.39, 3rd Ed.
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In plants, fungi, C. elegans Drosophila, a
RNA-dependent RNA polymerase (RDR) is involved in
the initiation (b) or amplification (c) of
silencing (RNAi).
CBP and PABP block access for RDR.
PABP missing.
D. Baulcombe 2004 Nature 431356
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Why RNAi 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 - first one identified, found in plant
  potyviruses (V. Vance)
• P19 - tomato bushy stunt virus, binds to siRNAs
  and prevents RISC formation (D. Baulcombe).
• Tat - RNA-binding protein from 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