1. RNA Interference Hannon, Nature 418:244-251
Jacques et al, Nature 418:435-8
Carmichael Nature 418:
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; 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

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 (RNA-induced silencing complex)
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. Differential RNAi requirements
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
sum fig. 4 here too.(stRNAs encoded by let-7 and lin-4

12. Modulation of HIV-1 replication by RNA interference
Jean-Marc Jacque, Karine Triques & Mario Stevenson
Nature Vol 418 p
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
siRNAs, b) mismatch siRNAs c) check on PKR pathway d) RT-PCR data
e) data from primary PBL

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-2a so that virus can not use translation machinery

15. Genomic HIV-1 RNA in NUCLEO-PROTEIN COMPLEXES is subject to specific RNAi degradation
a)exp. b)siRNAs did NOT affect viral infect. c) ???
d) effect of siRNAs of siRNAs of genomic viral RNA
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. Small RNAs Correspond to Centromere Heterochromatic Repeats
Science Vol. 297 Sept. 13, 2002
RNAi and Heterochromatin – a Hushed-Up Affair R. Allshire 297:
Regulation of Heterochromatic Silencing and Histone H3 Lysine-9 Methylation by RNAi
Volpe et al 297:
Small RNAs Correspond to Centromere Heterochromatic Repeats
Reinhart & Bartel 297:1831

18. TWO UNRELATED PATHWAYS???????
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.
*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.

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:

24. Jenuwein, T Science 297: