from Wu and Morris, Curr.Opin.Genet.Dev. 9, 237 (1999)

1
Examples of Homology-dependent Gene Silencing
TGS Pairing of tightly linked homologous loci
induces methylation
PTGS Transcript-specific degradation
SAS Spread of PTGS
RIP Induction of C-T transitions
RNAi
from Wu and Morris, Curr.Opin.Genet.Dev. 9, 237
(1999)
2
Models for Transvection
Transvection is an alteration of gene function
by homologous pairing
trans action of an element on a paired homolog
Propagation of chromatin structure to a paired
homolog
Pairing-sensitive silencing that acts at the
level of chromatin
RNA triggers silencing at paired homologs
Pairing of dissimilar homologs causes a
topological change
from Wu and Morris, Curr.Opin.Genet.Dev. 9, 237
(1999)
3
Response of Mammalian Cells to Long dsRNA
Long dsRNA induces interferon response in
vertebrates
PKR phosphorylates eIF2a to inhibit translation
2-5-oligoadenylate synthase is induced, which
activates RNaseL and leads to nonspecific mRNA
degradation
siRNA does not invoke the interferon response
from McManus and Sharp, Nature Rev.Genet. 3, 737
(2002)
4
The lin-14 Mutant has an Altered Pattern of Cell
Division
The PNDB neuroblast is generated prematurely
The LIN-14 protein prevents L2-type cell
divisions
from Lodish et al., Molecular Cell Biology, 6th
ed. Fig 21-6
5
miRNAs Regulate Development in C. elegans
The LIN-14 protein prevents L2-type cell
divisions
During L2, lin-4 miRNA prevents translation of
lin-14 mRNA
In the adult, let-7 inhibits lin-14 and lin-41
translation
Absence of LIN-41 permits lin-29 translation and
generation of adult cell lineages
from Lodish et al., Molecular Cell Biology, 6th
ed. Fig 21-6
6
lin-4 Inhibits Translation of lin-14 mRNA
Mutations in lin-4 disrupt regulation of larval
development in C. elegans
lin-4 antagonizes lin-14 function
lin-4 encodes a 22 nt-long microRNA that is
partially complementary to sites in the 3UTR of
lin-14 mRNA
Annealing of lin-4 to lin-14 mRNA inhibits
translation
from Li and Hannon, Nature Rev.Genet. 5, 522
(2004)
7
Biogenesis of miRNAs and siRNAs
miRNAs are genomically encoded
siRNAs are produced exogenously or from
bidirectionally transcribed RNAs
Drosha processes pri-miRNA to pre-miRNA in the
nucleus
miRNA is selectively incorporated into the RISC
for target recognition
Guide strand of siRNA is incorporated into the
RISC for target recognition
miRNAs have imperfect complementarity to their
target mRNA and inhibit translation
siRNAs form perfect duplex with their target
mRNA and trigger mRNA degradation
from Li and Hannon, Nature Rev.Genet. 5, 522
(2004)
8
Triggers of RNAi-Mediated Gene Silencing in
Mammals
from Mittal, Nature Rev.Genet. 5, 355 (2004)
9
Generation of miRNAs in Plants and Animals
In plants, miRNA maturation occurs in the nucleus
In animals, pre-miRNA is formed in the nucleus
and mature miRNA occurs in the cytoplasm
from Chen and Rajewsky, Nature Rev.Genet. 8, 93
(2007)
10
Strand Selection Into the RISC
The strand with its 5-terminus at the less
stable end of the duplex is incorporated into
the RISC
from Sontheimer, Nature Rev.Mol.Cell Biol. 6, 127
(2005)
11
Domain Structures of Dicer Enzymes
Dicer generates mature miRNA and siRNA in the
cytoplasm
In Arabidopsis, DCL-1 contains NLS and processes
pri-miRNA
from Li and Hannon, Nature Rev.Genet. 5, 522
(2004)
12
Strand Selection of Processed siRNA into the RISC
The PAZ domain of Dicer binds to the
pre-existing dsRNA end
The strand that has its 3-end bound to the PAZ
domain preferentially assembles into the RISC
from Sontheimer, Nature Rev.Mol.Cell Biol. 6, 127
(2005)
13
Guide RNA Loading Onto Argonaute
PAZ domain binds 3-overhang
5-end of guide RNA is anchored in a conserved
pocket of the PIWI domain
Argonaute slices passenger strand of siRNA
from Parker and Barford, Trends Biochem.Sci. 31,
622 (2006)
14
The Fate of mRNA Loaded With the miRISC
Targeted mRNA accumulates in P bodies
mRNA is stored in P bodies, undergoes
degradation, or reenters the translation pathway
from Rana, Nature Rev.Mol.Cell Biol. 8, 23 (2007)
15
Overview of RNA-Mediated Gene Silencing
siRNA
siRNA triggers endonucleolytic cleavage of
perfectly-matched complementary targets
Cleavage is catalyzes by Argonaute proteins
The resulting mRNA fragments are degraded
miRNA
miRNA triggers accelerated deadenylation and
decapping of partially-complementary targets
and requires Argonaute proteins and a P-body
component
miRNA represses translation
from Eulalio et al., Nature Rev.Mol.Cell Biol. 8,
9 (2007)
16
Regulation of siRNA Levels in C. elegans
RNA-dependent RNA polymerase amplifies siRNA
RRF-3 prevents siRNA amplification
ERI-1 is an siRNA-specific RNase
from Timmons, BioEssays 26, 715 (2004)
17
Prevalence of and Regulation by miRNAs
At least 800 miRNA-encoding genes in humans
At least 5000 genes are regulated by miRNAs
18
Human Accelerated Regions
HAR1-HAR49 are sequences that are highly
conserved among mammals, but have exhibited
rapid, recent sequence divergence
Most HARs occur outside protein coding regions
HAR1F RNA can fold into a stable stem-loop
secondary structure
HAR1F is expressed in the developing neocortex, a
region important in directing brain development
and neuronal migration
from Pollard et al., Nature 443, 167 (2006)
19
The Role of miRNA in Cancer
miRNA profiles define the cancer type better than
expression data from 16,000 mRNAs
miRNA expression is lower in cancers than in most
normal tissues
Down-regulation of all miRNAs enhanced tumor
growth
The undifferentiated state of malignant cells is
correlated with a decrease in miRNA expression
c13orf25 miRNA is the first non-coding oncogene,
is upregulated by c-Myc, and is involved in
leukemia development
c13orf25 inhibits expression of E2F1, a cell
cycle regulator
from He et al., Nature 435, 828 (2005)
Lu et al., Nature 435, 834 (2005)
20
miRNAs Suppress Breast Cancer Metastasis
Loss of miR-126 and miR-355 when human breast
cancer cells develop metastatic potential
Restoring expression of these miRNAs in malignant
cells suppresses metastasis in vivo
miR-355 targets the progenitor cell transcription
factor, SOX4, and the ECM component, tenascin C
from Tavasoie et al., Nature 451, 147 (2008)
21
A MicroRNA is Involved in Metastasis
Twist induces miR-10b transcription
miR-10b inhibits HOXD10 translation which
increases RHOC expression
miR-10b levels are elevated in
metastasis-positive patients
from Steeg, Nature 449, 671 (2007)
22
Immunostimulatory Effects of dsRNA
Long dsRNA induces PKR
Toll-like receptors in endosomes recognize dsRNA
and activate the interferon response
Blunt-ended dsRNA are recognized by RIG-1
helicase and activates the immune response
from Kim and Rossi, Nature Rev.Genet. 8, 173
(2007)
23
DNA Vector-based RNAi
from Shi, Trends Genet. 19, 9 (2003)
24
The Design of Optimal siRNAs
21 nt RNA that contains 2 nt 3- overhangs and
phosphorylated 5-ends
Lower stability at the 5-end of the antisense
terminus
Low stability in the RISC cleavage site
Low secondary structure in the targeted region
of the mRNA
from Mittal, Nature Rev.Genet. 5, 355 (2004)
25
Microarray-based Screening for Effective siRNA
Transfer a mixture with siRNA, target mRNA fused
with EGFP, and control RFP construct to a glass
slide
Overlay with a cell monolayer and transfect
Effective siRNA suppresses EGFP expression, but
not RFP expression
from Mittal, Nature Rev.Genet. 5, 355 (2004)
26
Delivery of siRNA for Therapy
siRNA is not taken up by most mammalian cells
Cholesterol-conjugated siRNA is taken up by the
LDL receptor
siRNA bound to targeted antibody linked to
protamine can achieve cell-specific siRNA
delivery
from Dykxhoorn and Lieberman, Cell 126, 231 (2006)
27
Cell-Specific Delivery of siRNA
Fuse Fab targeting antibody with protamine
siRNA binds noncovalently with protamine
Complex is endocytosed into cells expressing the
epitope
siRNA is released from the endosome and enters
the RISC
from Rossi et al., Nature Biotechnol. 23, 682
(2005)
28
Alphavirus-mediated RNAi
DNA inserted into an infectious cDNA silences
genes homologous to the insert
Dengue virus-resistant mosquitoes are produced by
inoculation of Aedes aegypti with dsSIN viruses
with Dengue virus inserts
Hairpin Dengue virus-specific RNA transcribed
from a plasmid generated virus-resistant cells
Induction of RNAi pathway in the midgut prior to
viral infection Production of genetically
modified mosquitoes that transcribe
virus-specific dsRNA in response to a blood
meal Potential to change vector competence
29
RNAi-dependent Chromatin Silencing in S. pombe
Overlapping RNAs from centromeric region is
processed into siRNA
siRNA activates or recruits Clr3
methyltransferase that methylates H3 on K9
Deletion of RNAi pathway genes cause loss of
silencing at centromeres and reduced H3 K9
methylation at centromeric regions
from Allshire, Science 297, 1818 (2002)
30
Small RNAs Modulate Viral Infection
Viral-encoded miRNA facilitate viral infection
and persistence
Host cell-encoded miRNAs inhibit or facilitate
viral replication
Viral suppressors of RNA silencing (VSR) inhibit
the RNAi pathway
31
Function of SV40 miRNA
SV40 miRNA is synthesized late in the viral life
cycle and targets TAg mRNA
SV40 miRNA aids immune invasion by reducing
susceptibility to lysis by CTLs
from Sarnow et al., Nature Reviews Microbiol. 4,
651 (2006)
32
Effects of Adenovirus VA1 MicroRNA
VA1 binds to and prevents PKR activation to
inhibit the innate immune response
VA1 competes with exportin-5 and inhibits Dicer
to inhibit the RNAi pathway
from Sarnow et al., Nature Reviews Microbiol. 4,
651 (2006)
33
A MicroRNA Protects HSV-1-infected Neurons from
Apoptosis
LAT is the only viral gene expressed during
latent infection in neurons
miR-LAT is generated from the LAT gene
miR-LAT downregulates TGF-b and SMAD3 and
contributes to the persistence of HSV-1 in
neurons in a latent form
from Gupta et al., Nature 442, 82 (2006)
Paper retracted 2008. Repeatedly unable to
detect miRNA
34
Cellular miRNAs Modulates Viral Infection
PFV-1 replication is stimulated by a plant VSR
implicating the role of small RNAs in the viral
life cycle
miR-32 inhibits viral replication
Tas is a PFV-1-encoded protein that prevents RNAi
miR-122 increases HCV replication in the liver
from Sarnow et al., Nature Reviews Microbiol. 4,
651 (2006)
35
Features of piRNAs
Piwi and Aubergine complexes contain piRNAs
antisense to transposon mRNAs
Argonaute3 complexes contain piRNAs biased to
the sense strand of transposon mRNAs
piRNAs display 10 nt complementarity at their
5-ends
from Aravin et al., Science 318, 761 (2007)
36
Model for Biogenesis of piRNAs that Target Mobile
Elements
Pool of piRNAs bind to Piwi or Aubergine anneals
to transposon mRNA target
Cleave transposon mRNA 10 nt from 5-end of
associated piRNA to create 5-end of Ago3 piRNA
Ago3-associated piRNA anneals to piRNA cluster
transcript to create additional copies of
antisense piRNA
Transposon is silenced
from Aravin et al., Science 318, 761 (2007)
37
(No Transcript)
38
Non-specific siRNA Inhibition of Angiogenesis
siRNAs targeting VEGF and VERGR1 are effective
treatments for macular degeneration
Non-specific siRNAs are also effective
siRNA inhibits angiogenesis by activating the
TLR3 signalling cascade
from Kalluri and Kanasaki, Nature 452, 543 (2008)
39
A MicroRNA Regulates Neuronal Differentiation
from Makeyev and Maniatis, Science 319, 1789
(2008)
40
Organismal Complexity May Be Due to Differences
in Regulation of Gene Expression
Number of protein-coding genes are similar in
animals
miRNA complexity correlates with an increase in
morphological complexity
from Technau, Nature 455, 1184 (2008)
41
Role of MicroRNAs and Epigenetics in Cancer
EZH2 overexpression promotes cell proliferation
Expression of EXH2 is inhibited by miR-101
miR-101 expression decreases during prostate
cancer progression
from Varambally et al., Science 322, 1695 (2008)