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. phenotypegene expressionDNA

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 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

PTGS (RNAi) occurs in wide variety of Eukaryotes: – Angiosperms –Chlamydomonas (unicellular) –Mammalian cells –C. elegans (nematode) –Drosophila –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 nt fragments, and in higher eukaryotes also pulls the strands apart via intrinsic helicase activity.

Model for RNAi By “Dicer” nt RNAs Fig , 3 rd 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.

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: Location in genome:intergenic regionsIntergenic regions, introns Clusters of miRNAs: Uncommon Common MiRNA biosynthesis:Dicer-like Drosha, Dicer Mechanism of repressionmRNA 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 genesRegulatory genes—crucial crucial for development, for development, structural enzymesproteins, enzymes