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Mammalian RNAi pathways Michael T. McManus MIT Center for Cancer Research what is RNA interference? •RNAi is a way to silence gene expression •to perform RNAi, dsRNA homologous to the targeted gene is made and then introduced into cells dsRNA nucleus •any mRNA with high sequence homology to the dsRNA may be silenced RNAi: a tool for inhibiting gene expression in vivo • • • • • • • • • C. elegans (Fire et al., 1998) Drosophila (Carthew et al., 1998) Planaria (Newmark et al., 1998) Trypanosomes (Ullu et al., 1998) Hydra (Lohmann et al., 1999) Zebrafish (Wargelius et al., 1999) Mice (Wianny & Zernicka-Goetz, 2000) “cosuppression” in plants “quelling” in Neurospora practical aspects of RNAi • biological research – defining gene function (gene knockout) • C. elegans genome RNAi projects – defining biochemical pathways • microarray screening of RNAi knockouts • therapeutic treatment – cancer – viral infection – parasitic infection How does RNAi work? RNAi works postranscriptionally…….. in key two steps! step one: processing the dsRNA into 21-23 nt fragments Qu ickT ime™ an d a GI F dec omp ress or are nee ded to se e this pic ture. 34 27 21 20 16 short-interfering RNA Tuschl, 2001 Dicer contains two RNAse III domains long dsRNA siRNAs siRNAs have a defined structure 19 nt duplex 2 nt 3’ overhangs step two: the antisense strand of the siRNA guides cleavage Tuschl, 2002 RNAi silencing complex • may be associated with translating ribosomes • active RNAse enzyme not yet identified • may participate in endogenous pathways that silence genes via translational repression Model for RNAi siRNA Mammals exhibit potent responses to dsRNA P P dsRNA PKR interferon production P eiF2a apoptosis Blockage of protein synthesis cell death smaller RNAs can escape the PKR pathway recall that siRNAs are intermediate effectors In the RNAi pathway siRNAs are not recognized by the PKR! need to further characterize mammalian RNAi how long does it last? how much dsRNA is required? can any region of a gene be effectively targeted? how to get siRNAs into the T-cells cationic lipids, calcium phosphate, etc. dead cells T-cell receptor-dependent transport, endocytosis, etc. no silencing electroporation develop an assay quantitative on the single-cell level CD8 T-cell CD4 flow cytometry detector fluorescent antibodies detect expression on the single cell level transfection of plasmids and siRNAs McManus, 2002 can any region of the mRNA be targeted with siRNAs? CD8 mRNA 5’ UTR CD8 ORF 3’ UTR CD8 siRNAs + CD4 mRNA 5’ UTR CD4 ORF CD4 siRNAs 3’ UTR + McManus, 2002 Influenza mRNA target-sites 5’ 3’ PB2 PB1 PA NP M NS UTR Coding sequence siRNA No inhibition Partial inhibition Strong inhibition Ge, 2003 how long does the RNAi response last? % cells silencing CD8 120.00 100.00 80.00 60.00 40.00 20.00 0.00 0 10 1000 100000 cell mass McManus, 2002 miniconclusion RNAi works by target degradation of the mRNA RNAi creates knock-downs, not knockouts! not every siRNA works RNAi via siRNAs is transient, lasting ~3-6 cell doublings establishing long-term RNAi Let the cell make the siRNA for you! CD8 hairpin RNAs McManus, 2002 hairpin siRNAs McManus, 2002 stable mammalian RNAi Within a three month window: Brummelkamp et al: Science Yu et al: PNAS Miyagishi et al: Nature Biotech Sui et al: PNAS Sook Lee et al: Nature Biotech Zeng et al: Mol Cell Paddison et al: Genes Dev Paul et al: Nature Biotech McManus et al:RNA lentiviral construct for siRNAs Rubinson et al Nature Genetics, 2003 Lentiviral CD8 knockdown Rubinson et al Nature Genetics, 2003 stable 14-fold CD8 knockdown by lentivirus siRNAs Rubinson et al Nature Genetics, 2003 functional silencing of genes in ES cell-derived mice by lentivirus-induced RNAi Rubinson et al Nature Genetics, 2003 mini-conclusion Although silencing by siRNAs is transient, vectors can be made to express siRNAs in cells RNAi knock-down mice can be generated in <30 days RNAi silencing can be transmitted through the germline