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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 posttranscriptional 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 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
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
Experimental use of RNAi
Possibly to fight viral infections???
• RNA interference can be used to posttranscriptionally 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 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
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/SGS2
Neurosp. QDE-1
C.elegans germline EGO-1
soma – RRF-1/RDE-9
Hypoth on amplification
Fig. 3 Hannon Review
Genetic Studies in C. elegans
RNAi silencing is heritable (unlike flies and mammals)
Differential RNAi
Possibly RDE-1 –4 are
requirements
required to deliver
exogenous dsRNA to
Parent
Dicer
requires RDE-1 –4
RDE-4 s dsRNA bind. prot.
Secondarily generated
both can interact w/ Dicer
dsRNA synthesized
from RdRP may need
another protein or
F1 progeny
exist in a complex w/
requires MUT-7 & RDE-2
RdRP and Dicer
sid-1 gene encodes
Many Models/ Hypoth.
transmembrane protein
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 EIF2a 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
Science Vol. 297 Sept. 13, 2002
RNAi and Heterochromatin – a
Hushed-Up Affair
R. Allshire
297:1818-1819
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
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