UT BIO 350M - Post transcriptional Gene Silencing

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Post-Transcriptional Gene Silencing (PTGS)Slide 2Antisense TechnologyDiscovery of PTGSDiscovery of PTGS (cont.)Slide 6Slide 7Slide 8Slide 9PTGS (RNAi) occurs in wide variety of Eukaryotes:Mechanism of RNAi: Role of DicerSlide 12Slide 13Why RNAi silencing?Micro RNAs (MiRNAs)Slide 16Slide 17Post-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 phenomenonEpigenetics - 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 compoundsDFR 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 geneFlowers 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 probeConclusions: (1) dsRNA reduced mex-3 mRNA better than antisense mRNA. (2) the suppressing signal moved from cell to cell. Fig. 16.29PTGS (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 Dicer1. 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.Model for RNAiBy “Dicer” 21-23 nt RNAsFig. 16.39, 3rd Ed.ATP-dependentHelicase or DicerActive siRNA complexes = RISC - contain Argonaute instead of DicerVery 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 431:356Why 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 HIVMicro 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 mutantComparison of Mechanisms of MiRNA Biogenesis and Action Better complementarity of MiRNAs and targets in plants.Summary of differences between plant and animal MiRNA systemsPlants Animals# of miRNA genes: 100-200 100-500Location in genome: intergenic regions Intergenic regions, intronsClusters of miRNAs: Uncommon CommonMiRNA biosynthesis: Dicer-like Drosha, DicerMechanism of repression mRNA cleavage Translational repressionLocation 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 multipleFunctions of known target genes: Regulatory genes Regulatory genes—crucialcrucial for development, for development, structural enzymes proteins,


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