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. • 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.
3. 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.
4. 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)
5. 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.
7. 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.
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 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
Conclusions: (1) dsRNA reduced mex-3 mRNA better than antisense
mRNA. (2) the suppressing signal moved from cell to cell.
Fig. 16.29
10. PTGS (RNAi) occurs in wide variety
of Eukaryotes:
– Angiosperms
– C. elegans (nematode)
– Drosophila
– Mammalian cells
– Chlamydomonas (unicellular
– Neurospora, but not in Yeast!
11. Mechanism of RNAi: Role of
Dicer
1. Cells (plants and animals) undergoing RNAi
contained small fragments (~25 nt) of the RNA being
suppressed.
2. A nuclease (Dicer) was purified from Drosophila
embryos that still had small RNA fragments
associated with it, both sense and antisense.
3. 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.
12. Generation of 21-23 nt fragments of target RNA in a
RNAi-competent Droso. embryo lysate/extract.
32
P-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
32
P-labeled, capped,
antisense Rr-luc RNA.
Fig. 16.30
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 32
P-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
14. Model for RNAi
By “Dicer”
21-23 nt RNAs
Fig. 16.39, 3rd
Ed.
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.
15. 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 431:356
16. 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
17. 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.
18. DCL1 mutant
Comparison of Mechanisms of MiRNA Biogenesis and Action
Better complementarity of MiRNAs and targets in plants.
19. 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 genes—crucial
crucial for development, for development, structural
enzymes proteins, enzymes
#7: These results resembled more what you get with antisense DFR genes, which suppress the endogenous DFR gene.
#9:Figure from Molec. Biology by Weaver. Work of Fire and Mello (2006 Nobel Prize)
#12:Philip Zamore et al., - dsRNA is cleaved independent of the presence of the corresponding mRNA.
#14:Dicer from higher eukaryotes has helicase activity, but not the small protein from Giardia, which was crystallized.
Active Si RNA complexes, or RISC, contain the nuclease Argonaute and not Dicer
#15:Baulcombe, D. (2004) RNA silencing in plants. Nature 431: 356-363.
#16:HCPro - Vicki Vance (not to be confused with Vicki Vale, Batman’s girlfriend)
P19 – D. Baulcombe
#18:DCL– dicer-like nuclease in plants; Drosha-nuclease that process pre-MiRNAs in animals.
MicroRNAs can inhibit binding of a translation initiation factor in animals.
#19:Millar, A.A. and P.M. Waterhouse (2005) Plant and animal microRNAs: similarities and differences. Functional & Integrative Genomics 5: 129-135
