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 any mRNA with high sequence homology to the dsRNA may be silenced nucleus dsRNA

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!

processing the dsRNA into nt fragments short-interfering RNA step one: Tuschl, 2001

Dicer contains two RNAse III domains siRNAs long dsRNA

siRNAs have a defined structure 19 nt duplex 2 nt 3 ’ overhangs

Tuschl, 2002 the antisense strand of the siRNA guides cleavage step two:

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

siRNA Model for RNAi

Mammals exhibit potent responses to dsRNA PKR P P P eiF2  dsRNA Blockage of protein synthesis interferon production cell death apoptosis

smaller RNAs can escape the PKR pathway siRNAs are not recognized by the PKR! recall that siRNAs are intermediate effectors In the RNAi pathway

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?

T-cell how to get siRNAs into the T-cells electroporation receptor-dependent transport, endocytosis, etc. cationic lipids, calcium phosphate, etc. dead cells no silencing

T-cell CD4 CD8 flow cytometry detector fluorescent antibodies detect expression on the single cell level develop an assay quantitative on the single-cell level

transfection of plasmids and siRNAs McManus, 2002

5’ UTR CD8 ORF3’ UTR CD8 siRNAs CD8 mRNA 5’ UTR CD4 ORF3’ UTR CD4 mRNA CD4 siRNAs can any region of the mRNA be targeted with siRNAs? + + McManus, 2002

PB2 PB1 PA NP M NS UTR 5’3’ Coding sequence No inhibition Partial inhibition Strong inhibition siRNA Influenza mRNA target-sites Ge, 2003

how long does the RNAi response last? cell mass % cells silencing CD8 McManus, 2002

miniconclusion RNAi via siRNAs is transient, lasting ~3-6 cell doublings RNAi creates knock-downs, not knockouts! not every siRNA works RNAi works by target degradation of the mRNA

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: McManus et al:RNA Brummelkamp et al: Science Paul et al: Nature Biotech Sui et al: PNAS Yu et al: PNAS Sook Lee et al: Nature Biotech Miyagishi et al: Nature Biotech Paddison et al: Genes Dev Zeng et al: Mol Cell

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 RNAi knock-down mice can be generated in <30 days RNAi silencing can be transmitted through the germline Although silencing by siRNAs is transient, vectors can be made to express siRNAs in cells