RNA Interference: A Natural Mechanism for Gene Silencing

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

2. 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

3. 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)

4. 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

5. 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

6. 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; dont 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

7. 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 ( R NA- i nduced s ilencing c omplex) Latent RISC w/ ds siRNAs +ATP Active RISC w/ ss siRNAs destroys target mRNAs Fig. 2. Hannon Review

8. 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

9. 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

10. 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.

11. Fig. 5 Hannon Review Model for the Mechanism of RNAi

12. 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

13. 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

14. 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- 2 so that virus can not use translation machinery

15. Genomic HIV-1 RNA in NUCLEO-PROTEIN COMPLEXES is subject to specific RNAi degradation Fig.2 HiV paper

16. 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

17. 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 RNAi and Heterochromatin a Hushed-Up Affair R. Allshire 297:1818-1819 Science Vol. 297 Sept. 13, 2002

18. 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.

19. 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

20. 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

21. 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

22. 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

23. 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

24. Jenuwein, T Science 297:2215-2218