RNA Interference

Hannon, Nature 418:244-251Jacques et al, Nature 418:435-8Carmichael 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 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; 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 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 +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:

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 gene

RdRP – RNA directed RNA polymerase, may be involved in amplification of signal; found in tomato

Arab. SDE1/SGS2Neurosp. QDE-1C.elegans germline EGO-1

soma – RRF-1/RDE-9Hypoth on amplification

Fig. 3 Hannon Review

Genetic Studies in C. elegansRNAi silencing is heritable (unlike flies and mammals)

Differential RNAi requirements

Parentrequires RDE-1 –4

RDE-4 s dsRNA bind. prot.both can interact w/ Dicer

F1 progenyrequires 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

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

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

Genomic HIV-1 RNA in

NUCLEO-PROTEIN COMPLEXES

is subject to specific RNAi degradation

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

Regulation of Heterochromatic Silencing and Histone H3 Lysine-9 Methylation by RNAi

Volpe et al 297:1833-1837Small RNAs Correspond to Centromere

Heterochromatic RepeatsReinhart & Bartel 297:1831

RNAi and Heterochromatin – a Hushed-Up Affair

R. Allshire 297:1818-1819

Science 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 expression

TWO 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 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