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

2
Discovery of PTGS

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

3
Discovery of PTGS

• First discovered in plants (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

4
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.

5
Flavonoid/anthocyanin pathway in plants
Strongly pigmented compounds
6
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 still doing
transcription!
7
Antisense Technology

• Antisense technology has been used for gt 20
  years.
• Based on introducing an antisense gene (or
  antisense RNA) into cells 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.
• The antisense effect was probably due to RNAi
  rather than inhibiting translation.

8

• 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 (and antisense) RNAs were contaminated
  with dsRNA.
• dsRNA was the suppressing agent.

9
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. 16.37
10
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 budding yeast (S.
  cerevisiae)!

11
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.

12
Fig. 16.38
Generation of 21-23 nt fragments of target RNA in
a RNAi-competent Drosophila embryo 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.
13
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. 16.39
14
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. There is an exceptional cleavage only
9 nt away from the adjacent cleavage (induced by
dsRNA C) this site had a stretch of 7 Us. 14 of
16 cleavage sites were at a U.
Conclusion Dicer cleaves at 23-nt intervals
after U.
15
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.
16
In Plants, fungi, C. elegans, 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
17
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

18
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.
20
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-clea
vage 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.