RNA Interference

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RNA Interference

• Hannon, Nature 418244-251
• Jacques et al, Nature 418435-8
• Carmichael Nature 418379-380
• Allshire, Science 2971818-9

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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

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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)

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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

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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
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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 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

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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
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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

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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
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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.

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

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PKR (RNA-dependent protein kinase)
PathwayNon-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

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Genomic HIV-1 RNA in NUCLEO-PROTEIN COMPLEXESis
subject to specific RNAi degradation
Fig.2 HiV paper
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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 20-30x

Fig. 3 HIV Paper
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RNAi and Heterochromatin a Hushed-Up AffairR.
Allshire 2971818-1819
Science Vol. 297 Sept. 13, 2002

• Regulation of Heterochromatic Silencing and
  Histone H3 Lysine-9 Methylation by RNAi
• Volpe et al 2971833-1837
• Small RNAs Correspond to Centromere
  Heterochromatic Repeats
• Reinhart Bartel 2971831

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• 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.

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• 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

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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

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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

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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

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• 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 2971833-1837

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Jenuwein, T Science 2972215-2218