Gene Silencing (2) PDF

Molecular Biotechnology BE618 Lecture 7 Regulation of gene expression Gene silencing © Prof. Subramanian Sankaranarayanan Post-Transcriptional Gene Silencing (PTGS) Also called RNA interference or RNAi Process results in down-regulation of a gene at the RNA level...

Molecular Biotechnology BE618 Lecture 7 Regulation of gene expression Gene silencing © Prof. Subramanian Sankaranarayanan 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 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 “Discoveries cannot be planned, they pop up, like puck, in unexpected corners- Max Perutz” 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. Sir David Baulcombe  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. (Mex-3 Muscle protein) 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 Chlamydomonas (unicellular) Mammalian cells C. elegans (nematode) Drosophila Neurospora, but not in Yeast! https://www.umassmed.edu/rti/ biology/rna/how-rnai-works/ 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.  Model for RNAi By “Dicer” 21-23 nt RNAs ATP-dependent Helicase or Dicer Very efficient process Active because many small siRNA interfering RNAs complexes (siRNAs) generated = RISC from a larger dsRNA. - contain Argonaute instead of Dicer Fig. 16.39, 3rd Ed.  RNAi mechanism https://www.youtube.com/watch?v=cK-OGB1_ELE  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. Discovery of microRNA in 1993 and Nobel prize in 2024 (31 years!) Victor Ambros and Gary Ruvkun  In the late 1980s, Victor Ambros and Gary Ruvkun were postdoctoral fellows in the laboratory of Robert Horvitz, who was awarded the Nobel Prize in 2002, In Horvitz’s laboratory, they studied a relatively unassuming 1 mm long roundworm, C. elegans. They studied two mutant strains of worms, lin-4 and lin- 14, that displayed defects in the timing of activation of genetic programs during development. https://www.nobelprize.org/prizes/medicine/2024/ press-release/ A Comprehensive Review of Small Interfering RNAs (siRNAs): Mechanism, Therapeutic Targ ets, and Delivery Strategies for Cancer Thera https://geneticeducation.co.in/sirna-vs-mirna-10- major-differences/  GLO1 is essential for successful pollination Wes R1 R2 R3 MG-modified proteins 52 kDa CBB IB: anti-MG RNAi mediated suppression of GLO1 in stigmas results plants with reduced seed set and concomitant increase in Methylgyloxal levels Sankaranarayanan et al., 2015 11 Transgenic GLO1 RNAi lines displayed reduced pollen attachment and germination * * 12 Comparison of Mechanisms of MiRNA Biogenesis and Action DCL1 mutant DICER-LIKE1 (DCL 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