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 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 Silencing
Dicer 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:
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/SGS2Neurosp. QDE-1C.elegans germline EGO-1
soma – RRF-1/RDE-9
Hypoth on amplificationFig. 3 Hannon Review
Genetic Studies in C. elegansRNAi 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
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 297:1833-1837
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
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.
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