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
• 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.
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.
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)
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.
Flavonoid/anthocyanin pathway in plants
Strongly pigmented compounds
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.
• 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.
Double-stranded RNA (dsRNA) induced
interference of the Mex-3 mRNA in the nematode
C. elegans.
Antisense RNA (c) or
dsRNA (d) for the mex3 (mRNA) was injected
into C. elegans
ovaries, and then mex3 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
PTGS (RNAi) occurs in wide variety
of Eukaryotes:
– Angiosperms
– C. elegans (nematode)
– Drosophila
– Mammalian cells
– Chlamydomonas (unicellular
– Neurospora, but not in Yeast!
Mechanism of RNAi: Role of
Dicer
1.
2.
3.
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.
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.
Fig. 16.30
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
Model for RNAi
By “Dicer”
21-23 nt RNAs
ATP-dependent
Helicase or Dicer
Very efficient process
because many small
interfering RNAs
(siRNAs) generated
from a larger dsRNA.
Fig. 16.39, 3rd Ed.
Active
siRNA
complexes
= RISC
- contain
Argonaute
instead of
Dicer
In plants, fungi, C. elegans & Drosophila, a RNAdependent 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 431:356
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:
1. HCPro - first one identified, found in plant
potyviruses (V. Vance)
2. P19 - tomato bushy stunt virus, binds to
siRNAs and prevents RISC formation
(D. Baulcombe).
3. Tat - RNA-binding protein from HIV
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.
Comparison of Mechanisms of MiRNA Biogenesis and Action
DCL1 mutant
Better complementarity of MiRNAs and targets in plants.
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
the open-reading frame
Predominantly the 3′-UTR
Generally one
Generally multiple
Regulatory genes
crucial for development,
enzymes
Regulatory genes—crucial
for development, structural
proteins, enzymes
# of miRNA binding
sites in a target gene:
Functions of known
target genes: