1 / 19

RNA interference (RNAi)

RNA interference (RNAi). white RNAi hairpin. Wild-type. A Brief History of RNAi. Hint 1995, Guo and Kemphues: Injection of par-1 gene sense RNA into the gonad of C. elegans induced par-1 null phenocopies at the same high frequency as injection of anti-sense RNA. First description

briar
Download Presentation

RNA interference (RNAi)

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. RNA interference (RNAi) white RNAi hairpin Wild-type

  2. A Brief History of RNAi • Hint • 1995, Guo and Kemphues: Injection of par-1 gene sense RNA into the gonad of • C. elegans induced par-1 null phenocopies at the same high frequency as injection • of anti-sense RNA. • First description • 1998, Fire et al.: Injection of dsRNA for specific genes into C. elegans caused • a specific disappearance of the gene products from somatic cells and F1 progeny • effect was on stability of mRNA • crossed cellular barriers • only a few molecules of dsRNA per cell required • dsRNA from exons but not introns had effect

  3. Brief History contd. • Mechanism • 1999, Hamilton and Baulcombe: Arabidopsis plants undergoing post-transcriptional gene silencing (PTGS) contained 21-25 nt long RNAs that were complementary to both strands of the silenced gene and that were processed from a long dsRNA precursor • 2000, Zamore et al. : Used Drosophila embryo extracts to show that long dsRNAs are processed to 21-23 nt RNAs that direct targeted mRNA cleavage • 2001, Bernstein et al.: Using Drosophila S2 cell extracts, these authors described the enzyme for producing the 21-23nt RNAs: an Rnase III enzyme, Dicer. • 21-23 nt RNAs = short interfering RNAs (“siRNAs”)

  4. siRNA Structure and Formation 5’ phosphate siRNA Dicer cleaves dsRNA siRNAs are incorporated into RISC (RNA Induced Silencing Complex) siRNAs unwind and guide RISC to a substrate mRNA substrate cleavage (From McManus and Sharp, 2002 & Hannon, 2002)

  5. Small temporal RNAs (stRNAs) • C. elegans let-7 and lin-4: • isolated as heterochronic mutants • negative regulators of specific protein-coding genes • (target the 3’ UTR) • encode small RNAs, synthesized as 70 nt precursors • post-transcriptionally processed to a 21 nt mature form • by Dicer • regulate expression at the level of translation • archetypes of a large class of endogenously encoded • small RNAs now called microRNAs (miRNAs)

  6. siRNAs vs stRNAs

  7. RNAi in worms • heritable • systemic • Methods: • injection of dsRNA into gonad • soak worms in dsRNA • feed worms E. coli transformed with a plasmid expressing • S and AS RNAs

  8. Large scale RNAi screen in worms Gonczy et al., 2000 • targeted 96% of ORFs on chromosome III • used PCR primers tailed with T3 and T7 promoter sequences • PCR product size: 500+ bp • product encompassed > 90% coding sequence

  9. RNAi in Flies • non-inheritable • cell autonomous • Methods: • injection of dsRNA into syncytial blastoderm embryos • (Kennerdell and Carthew, 1998) • observed variability in interference activities of • different dsRNAs (null phenotypes and mosaics) • phenotype localized to site of injection • Inducible RNAi transgenes • dsRNAs • Snap-back RNAi • extended hairpin loop RNAs • Genomic cDNA hybrids

  10. Examples and Results • 2000, Lam and Thummel: established P-element transformants • that use the heat-inducible hsp70 promoter to drive expression • of a snapback dsRNA corresponding to the coding region of EcR • and BFTZ-F1 • established 8 hs-EcRi and 3 hs-FF1i lines; had variable effects NotI BamH1 XbaI EcoR1 hsp70 Act 5C termination and polyA signals pCaSpeR-hs-act P-element vector

  11. Examples and Results contd. • 2000, Kennerdell and Carthew: expressed an extended hairpin-loop • loop RNA from a transgene • UAS-geneRNAi X different Gal4 strains • Kirby et al. 2002: Sod2 RNAi; made inverted-repeat structure of Sod2 • cDNA and cloned into EcoR1 site of pPUAST • generated 18 lines and analyzed 2 with “robust” expression • Leulier et al. 2002: dFADD RNAi; 500 bp long cDNA fragment • (nt positions 1-500 of coding seq) was amplified by PCR and inserted • as an inverted repeat (IR) into a modified Bluescript vector, pSC1, which • possesses an IR formation site consisting of paired CpoI and SfiI • RE sites; IRs in a head-to-head orientation; IR fragment cut out and • cloned into pUAST ; used at least 2 independent lines for each expt.

  12. Genomic-cDNA fusions • Kalidas and Smith, 2002: used genomic-cDNA fusion construct • regulated by its own promoter; pUAST system; 3 genes • Genomic fragment contains 5’UTR and intron + exon sequences • cDNA fragment contains corresponding exon sequences • two fragments are fused, head-to-head; apparently more stable • and easier to clone; splices to form mature • mRNA which then forms hairpin dsRNA • analyzed two independent lines for each construct • claim that suppression is greater and more uniform but no direct • comparison between methods

  13. RNAi in Drosophila cell cultures (Perrimon lab/RNAi Genome Project) 25 – 75 nM (0.2 ug) of 500 nt dsRNAs

  14. Functional genomic analysis of phagocytosis using Drosophila S2 cells and RNAi (Ramet et al. 2002) • generated random templates from an S2 cell-derived • cDNA library cloned into pcDNAI plasmids • Pooled two plasmids and generated S and AS RNA using • T7 and SP6 • 5 x 105 S2 cells were treated with 40ug dsRNA (20ug • per gene) for 60h and then analyzed • examined 1,000 random dsRNAs; found 34 genes with a • detectable effect on phagocytosis

  15. RNAi in mammals • RNAi used in early mouse embryos • BUT mammalian somatic cells exhibit a nonspecific response • to dsRNA • long dsRNA activates the RNA-dependent protein kinase • (PKR) pathway which phosphorylates EIF-2A and arrests • translation • synthetic siRNAs do not activate PKR (likely too small) and • can induce gene knockdowns

  16. RNAi in mammals contd. • siRNA and a lipophilic agent used to transfect cells • Limiting factor is the transfectability of cells; • HeLa cells are the cell line of choice • effects of siRNAs are transient since mammalian cells • lack amplification mechanisms • most recent experimental approach is modelled on miRNAs • short hairpin RNAs (shRNAs) are expressed in vivo from • DNA vectors containing RNA pol III promoters (H1, U6) • shown to induce stable suppression in mammalian cells

  17. Designing DNA silencing constructs Hairpin siRNA-in-trans (From McManus and Sharp, 2002)

  18. Use of DNA silencing constructs (From McManus and Sharp, 2002)

  19. Practical Considerations of siRNA design • select base-pairing region carefully to avoid chance • complementarity to an unrelated mRNA i.e. BLAST • N.B.: RNAi can tolerate siRNA:mRNA mismatches • of 1 – 2 bp • mRNA region optimal for siRNA targeting is not yet known; • suggested region is the first 50 – 100 nt of a cDNA • sequence, downstream of the translation start site • (want to avoid regulatory protein binding sites) • optimal design for shRNAs not yet known

More Related