210 likes | 409 Views
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
E N D
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 • 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
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”)
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)
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)
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
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
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
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
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.
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
RNAi in Drosophila cell cultures (Perrimon lab/RNAi Genome Project) 25 – 75 nM (0.2 ug) of 500 nt dsRNAs
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
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
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
Designing DNA silencing constructs Hairpin siRNA-in-trans (From McManus and Sharp, 2002)
Use of DNA silencing constructs (From McManus and Sharp, 2002)
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