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RNA interference (RNAi) Wild-typewhite RNAi hairpin.

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Presentation on theme: "RNA interference (RNAi) Wild-typewhite RNAi hairpin."— Presentation transcript:

1 RNA interference (RNAi) Wild-typewhite RNAi hairpin

2 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 A Brief History of RNAi

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

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 EcoR1 NotIXbaIBamH1 hsp70Act 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 10 5 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

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