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 petuniaCaMV - 35S promoter from Cauliflower Mosaic VirusDFR 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 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

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. 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.

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 RNAiBy “Dicer”

21-23 nt RNAs

Fig. 16.39, 3rd Ed.

ATP-dependentHelicase 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.

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

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.

DCL1 mutant

Comparison of Mechanisms of MiRNA Biogenesis and Action

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 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