RNA Interference Hannon, Nature 418:244-251 Jacques et al, Nature 418:435-8 Carmichael Nature 418:379-380 Allshire, Science 297:1818-9

RNA Interference (RNAi) Double stranded RNA responsible for post-transcriptional gene silencing of the gene from which it was derived. SPECIFIC NATURAL BIOLOGICAL MECHANISM IN PLANTS, INSECTS AND MAMMALS RNAi FUNCTIONS regulates expression of protein coding genes mediates resistance to both exogenous parasitic and exogenous pathogenic nucleic acid used experimentally to block gene expression

Historically Important Discoveries 1990 – exogenous transgenes in petunias caused variegated pigmentation (Co-suppression) Plant destruction of viral RNA; endogenous genes could be silenced if homologous sequences were present in the virus replicon Discovered (1998) in C. elegans –dsRNA response resulting in sequence-specific gene silencing SILENCEING dsRNA 10x greater than (+) or (-) sense RNA dsRNA induced gene silencing found in many euk. (Fig. 1)

Why RNAi? Hypotheses and Clues Include: RNAi mechanism evolved to immobilize transposable elements and silence RNA viruses ie Mut7 -/- C. elegans; has a mutator phenotype b/c transposable element Later RNAi important in silencing chromatin – may recruit Clr4 histone H3 methylase small RNAs have been correlated w/ methylation of promoter DNA of Arabidopsis (S.pombe has no DNA methylation) both siRNAs and miRNAs regulate gene expression

Exogenous and Endogenous RNAi Silencing Complex = ds siRNAi (21-23bp) Proteins* ie RISC Complexes recognize complementary ss mRNA Results in target mRNA cleavage; no protein product

Experimental use of RNAi Possibly to fight viral infections??? RNA interference can be used to post-transcriptionally silence or suppress a gene (CELLULAR or VIRAL) thru mRNA degradation; don’t need knock out mutants RNAi testing of C. elegans ~19,000 genes! Imagenex sells the “RNAi Gene Suppressor System” – a plasmid vector based RNAi system the allows suppression of genes in mammalian cells sRNAi too small to induce PKR Pathway

Mechanism of dsRNA Gene Silencing Dicer endonuclease enzyme dimer cleaves RNAi (RNAse III family) Small ~ 22 nucleotide RNAs assoc. w/ RISC (guide RNAs) Effector Nuclease = RISC (RNA-induced silencing complex) Latent RISC w/ ds siRNAs +ATP Active RISC w/ ss siRNAs – destroys target mRNAs Fig. 2. Hannon Review

RISC –nuclease complex Precursor RISC ~ 250K Active RISC ~100K (siRNA unwinding) RISC COMPONENTS: siRNA endonuclease Drosoph. work indicates exonuclease AGO2 protein (PAZ and PIWI domains) possibly involved in shuttling of siRNAs to RISC

Spreading and Amplification of Silencing Transitive RNAi – movement of silencing 3’ to 5’ along a gene RdRP – RNA directed RNA polymerase, may be involved in amplification of signal; found in tomato Arab. SDE1/SGS2 Neurosp. QDE-1 C.elegans germline EGO-1 soma – RRF-1/RDE-9 Hypoth on amplification Fig. 3 Hannon Review

Differential RNAi requirements Genetic Studies in C. elegans RNAi silencing is heritable (unlike flies and mammals) Differential RNAi requirements Parent requires RDE-1 –4 RDE-4 s dsRNA bind. prot. both can interact w/ Dicer F1 progeny requires MUT-7 & RDE-2 sid-1 gene encodes transmembrane protein Possibly RDE-1 –4 are required to deliver exogenous dsRNA to Dicer Secondarily generated dsRNA synthesized from RdRP may need another protein or exist in a complex w/ RdRP and Dicer Many Models/ Hypoth.

Fig. 5 Hannon Review – Model for the Mechanism of RNAi sum fig. 4 here too.(stRNAs encoded by let-7 and lin-4

Modulation of HIV-1 replication by RNA interference Jean-Marc Jacque, Karine Triques & Mario Stevenson Nature Vol 418 p. 435-438 Silencing viruses with RNA G.Carmichael

Introduced 22 nucleotide synthetic siRNAs (complementary to HIV target +/- GFP) into human cell lines/ primary lymphocytes RESULTS: DO NOT ACTIVATE PKR PATHWAY and siRNAs SPECIFICALLY DEGRADE HIV-1 mRNA, dsRNA-activated protein kinase siRNAs, b) mismatch siRNAs c) check on PKR pathway d) RT-PCR data e) data from primary PBL

PKR (RNA-dependent protein kinase) Pathway Non-specific dsRNA Response Mammalian anti-viral response; dsRNA viruses or viruses w/ dsRNA intermediates Host shut down of translation via Phosphorylation of EIF-2a so that virus can not use translation machinery

Genomic HIV-1 RNA in NUCLEO-PROTEIN COMPLEXES is subject to specific RNAi degradation a)exp. b)siRNAs did NOT affect viral infect. c) ??? d) effect of siRNAs of siRNAs of genomic viral RNA Fig.2 HiV paper

siRNAs from plasmid templates can inhibit HIV-1 Plasmid expression under T7 RNA pol. promoter of self-complementary RNA; results in dsRNA “hairpin” ALL suppressed viral production 20-30x Fig. 3 HIV Paper

Small RNAs Correspond to Centromere Heterochromatic Repeats Science Vol. 297 Sept. 13, 2002 RNAi and Heterochromatin – a Hushed-Up Affair R. Allshire 297:1818-1819 Regulation of Heterochromatic Silencing and Histone H3 Lysine-9 Methylation by RNAi Volpe et al 297:1833-1837 Small RNAs Correspond to Centromere Heterochromatic Repeats Reinhart & Bartel 297:1831

TWO UNRELATED PATHWAYS??????? Heterochromatin*-repetitive, condensed part of genome Post-translation modific. of histone tails important Transgenes inserted into heterochromatin are shut off SILENT CHROMATIN formed by DEACETYLATION and subsequent METHYLATION of Histone H3 Lys9 RNAi also affects silencing of gene expression TWO UNRELATED PATHWAYS??????? S. pombe (yeast) research finds that BOTH ARE PART OF THE SAME GENE-SILENCING PATH. *hetchrom. important in imprinting, dosage compensation, recombination, chromosome condensation Hetchrom. at centromeres is ESSENTIAL for accurate segregation of chromosomes during cell division; at telomeres hetchro. protects against degradation, and linking chromo.

in S. pombe repetitive DNA near centromeres is silenced via METHYLATION of H3 Lys9 and binding of Swi6 (gene express ON if Lys4 methylated) Volpe et al. found that deleting genes in RNAi pathway (argonaute, Dicer, Rdp1*) lead to LOSS of GENE SILENCING of transgenes inserted into heterochromatin RNAi and Heterochromatin Silencing are RELATED Pathways

How does the RNAi machinery aid in the formation of silent chromatin? Possibility that siRNAs bring methyltransferases to the target loci, where they are important in histone tail modification ie. Drosoph. targets acteyltransferase w/ RNA binding chromodomain to histone H4

siRNA and Silent Chromatin - Model RNA homologous to centromeric repeats are processed – siRNAs siRNAs may recruit Clr4 histone H3 methylase result in meth. of H3 Lys9 Swi6 binds chromatin Gene silencing

Related Gene Silencing Mechanisms May Function in Mammals X chromosome inactivation in mammals Xist RNA coating of inactive X chromosome, but no data yet suggests that Xist is processed by RNAi machinery *** Future work using RNAi introduced in experiments should include study of chromatin structure or modifications at the locus of the affected gene

Mouse – X inactivation and Igf2r imprinting are mediated by noncoding antisense RNA Possibly in organisms w/ DNA methylation; Histone protein modification similar to S. pombe would in turn cause DNA methylation and subsequent gene silencing regulation FOR MORE INFO. ON CORRELATION SEE Volpe et al. SCI 297:1833-1837

Jenuwein, T Science 297:2215-2218