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

RNA Interference (RNAi)Double stranded RNA responsible for post-transcriptional gene silencing of the gene from which it was derived. SPECIFICNATURAL BIOLOGICAL MECHANISM IN PLANTS, INSECTS AND MAMMALSRNAi FUNCTIONSregulates expression of protein coding genesmediates resistance to both exogenous parasitic and exogenous pathogenic nucleic acidused experimentally to block gene expression

Historically Important Discoveries1990 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 repliconDiscovered (1998) in C. elegans dsRNA response resulting in sequence-specific gene silencingSILENCEING dsRNA 10x greater than (+) or (-) sense RNAdsRNA induced gene silencing found in many euk. (Fig. 1)

Why RNAi?Hypotheses and Clues Include:RNAi mechanism evolved to immobilizetransposable elements and silence RNA virusesie Mut7 -/- C. elegans; has a mutator phenotype b/c transposable elementLater RNAi important in silencing chromatin may recruit Clr4 histone H3 methylasesmall 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 RNAiSilencing Complex = ds siRNAi (21-23bp) Proteins* ie RISC Complexes recognize complementary ss mRNA Results in target mRNA cleavage; no protein product

Experimental use of RNAiPossibly 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 mutantsRNAi 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 cellssRNAi too small to induce PKR Pathway

Mechanism of dsRNA Gene SilencingDicer endonuclease enzyme dimer cleaves RNAi (RNAse III family)Small ~ 22 nucleotide RNAsassoc. w/ RISC (guide RNAs)Effector Nuclease = RISC (RNA-induced silencing complex)Latent RISC w/ ds siRNAs +ATPActive RISC w/ ss siRNAs destroys target mRNAs

Fig. 2. Hannon Review

RISC nuclease complexPrecursor RISC ~ 250K Active RISC ~100K (siRNA unwinding)RISC COMPONENTS:siRNAendonucleaseDrosoph. work indicates exonucleaseAGO2 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 geneRdRP RNA directed RNA polymerase, may be involved in amplification of signal; found in tomatoArab. SDE1/SGS2Neurosp. QDE-1C.elegans germline EGO-1 soma RRF-1/RDE-9Hypoth on amplificationFig. 3 Hannon Review

Genetic Studies in C. elegansRNAi silencing is heritable (unlike flies and mammals)Differential RNAi requirementsParentrequires RDE-1 4 RDE-4 s dsRNA bind. prot.both can interact w/ Dicer

F1 progenyrequires MUT-7 & RDE-2sid-1 gene encodes transmembrane proteinPossibly 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 DicerMany Models/ Hypoth.

Fig. 5 Hannon Review Model for the Mechanism of RNAi

Modulation of HIV-1 replication by RNA interferenceJean-Marc Jacque, Karine Triques & Mario StevensonNature Vol 418 p. 435-438Silencing 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 PATHWAYand siRNAs SPECIFICALLY DEGRADE HIV-1 mRNA, dsRNA-activated protein kinase

PKR (RNA-dependent protein kinase) PathwayNon-specific dsRNA ResponseMammalian 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 COMPLEXESis subject to specific RNAi degradation Fig.2 HiV paper

siRNAs from plasmidtemplates can inhibit HIV-1

Plasmid expression under T7 RNA pol. promoter of self-complementary RNA; results in dsRNA hairpin

ALL suppressed viral production 20-30xFig. 3 HIV Paper

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

Heterochromatin*-repetitive, condensed part of genomePost-translation modific. of histone tails importantTransgenes inserted into heterochromatin are shut offSILENT CHROMATIN formed by DEACETYLATION and subsequentMETHYLATION of Histone H3 Lys9 RNAi also affects silencing of gene expressionTWO UNRELATED PATHWAYS???????S. pombe (yeast) research finds that BOTH ARE PART OF THE SAME GENE-SILENCING PATH.

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 modificationie. Drosoph. targets acteyltransferase w/ RNA binding chromodomain to histone H4

siRNA and Silent Chromatin - Model RNA homologous to centromeric repeats are processed siRNAssiRNAs may recruit Clr4 histone H3 methylase result in meth. of H3 Lys9Swi6 binds chromatin Gene silencing

Related Gene Silencing Mechanisms May Function in MammalsX chromosome inactivation in mammalsXist 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

sum fig. 4 here too.(stRNAs encoded by let-7 and lin-4siRNAs, b) mismatch siRNAs c) check on PKR pathway d) RT-PCR datae) data from primary PBLa)exp. b)siRNAs did NOT affect viral infect. c) ???d) effect of siRNAs of siRNAs of genomic viral RNA*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.