RNAi Mechanism

The Central Dogma DNA (double-stranded) RNA (single-stranded) Protein

When good RNA goes bad.... Viruses can make double- stranded RNA

When good RNA goes bad.... Cells sense double- stranded RNA and activate a response called RNAi

Understanding how RNAi works is the key to using it as a genetic tool and for therapy

Cell free extract RNAi? What is the mechanism of interference? RNAi in vitro... Hannon Lab, Zamore Lab, Tuschl Lab, Sharp Lab

Dicer cuts dsRNA into short RNAs Vanhecke, D.; Janitz, M. Drug Discov. Today, 2005, 10, Role for a bidentate ribonuclease in the initiation step of RNA interference Emily Bernstein, Amy A. Caudy, Scott M. Hammond & Gregory J. Hannon 2001 Bernstein et al. Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as two dsRNA-binding domains.

Dicer is an evolutionarily conserved nuclease

dsRNA RNAi functions in many different organisms

GFP – germline transgene Dicer is required for RNAi GFP dsRNA wild-type dcr1 -/-

How do small dsRNAs turn genes off?

Dicer-cut siRNA is used as a template to direct cleavage of complementary mRNA by RISC RISC: RNA-inducing silencing complex

Argonaute proteins lie at the heart of RISC Ji-Joon Song Leemor Joshua-Tor

Argonaute-PIWIRNase HIRNase HII PIWI is an RNase H like domain Ji Joon Song Leemor Joshua-Tor

A model for siRNA directed cleavage

microRNAs regulate gene expression without cleavage

small temporal RNAs (stRNAs) stRNA precursor stRNA Translational repression RNA or protein level L1 stageL2 stageL3 stageL4 stageAdult stage LIN-14, LIN-28 proteinsLIN-41 proteinLIN-29 protein let-7 RNA lin-4 RNA Ambros lab, Rukvun lab Dicer Mello lab, Zamore lab lin-4 miRNA represses translation of lin-14 (L1 to L2 moult) Banerjee & Slack, 2002

siRNA miRNA RNAi acts to regulate gene expression by two dicer-dependent mechanisms

Model for translational repression M7 GpppG AAAAAA RISC RISC recognizes a target

Model for translational repression M7 GpppG AAAAAA RISC DCP GW other Other proteins are also recruited – either along with RISC or later

Model for translational repression M7 GpppG AAAAAA RISC DCP GW other decapping to P-bodies block translation? (e.g. Filipowicz) de-adenylation? (e.g. Giraldez, Belasco, Rivas) RISC may block through multiple mechanisms

Least well understood - Arabidopsis as a model….

DNA is embedded in chromatin

Regulation of gene expression at the level of chromatin Sequence-independent linker histones: control DNA compaction and accessibility to trans-acting factors post-translational modifications of histone tails: control compaction of DNA and serve as docking sites for trans-acting factors Range: Can act at the level of a single gene, often acts over groups of genes and over larger domains (20-200kb), and can affect gene expression over an entire chromosome

Model for siRNA-dependent initiation of heterochromatic silencing by RITS RITS: RNA-induced initiation of transcriptional gene silencing complex

dsRNA-mediated silencing in various organisms: Multiple mechanisms that are Dicer-dependant Meister & Tuschl, 2004

Levine and Davidson, PNAS, 2005 Ke, et al. Current Opinion in Chemical Biology 2003 dsRNA-mediated regulation may play a central role in the regulation of Gene Networks