Transcript Nature Rev.Genet. 12

Examples of Homology-dependent Gene Silencing
TGS – Pairing of tightly linked
homologous loci induces methylation
Transcriptional Gene Silencing
PTGS – Transcript-specific degradation
Post-transcriptional Gene Silencing
SAS – Spread of PTGS
Systemic Acquired Silencing
RIP – Induction of C-T transitions
Repeat-induced Point Mutation
RNAi
RNA interference
from Wu and Morris, Curr.Opin.Genet.Dev. 9, 237 (1999)
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)
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)
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
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
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 3’UTR of lin-14 mRNA
Annealing of lin-4 to lin-14
mRNA inhibits translation
from Li and Hannon, Nature Rev.Genet. 5, 522 (2004)
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)
Triggers of RNAi-Mediated Gene Silencing in Mammals
from Mittal, Nature Rev.Genet. 5, 355 (2004)
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)
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)
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)
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)
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)
Mechanisms of miRNA Sequence Diversification
Seed shifting results from variations
in Drosha or Dicer processing
In arm shifting, mutations within
the precursor change the ratio
of miRNA to miRNA* loading
In hairpin shifting, the folding is
changed into a new configuration
In cells containing adenosine
deaminase, A is converted to I
from Berezikov, Nature Rev.Genet. 12, 846 (2011)
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)
Role of Poly(A) and Cap in Translation Initiation
The cap structure is recognized by eIF4F
Poly(A) is recognized by PABPC
PABPC interacts with eIF4G
Recruitment of the preinitiation
complex is increased
from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011)
miRNAs Promote mRNA Deadenylation
miRNA guide strand associates with AGO
AGO interacts with GW182
GW182 may compete with
eIF4G for binding to PABPC
and prevents mRNA circularization
GW182 may reduce the affinity
of PABPC for the poly(A) tail
Assembly of AGO-GW182-PABPC complex
triggers deadenylation by CAF1-CCR4-NOT
from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011)
Fate of Deadenylated mRNAs
Deadenylated mRNAs are stored
in a translationally repressed state
Deadenylated mRNAs are decapped by
DCP2 associated with decapping activators
Decapped mRNA is degraded by XRN1
from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011)
Overview of RNA-Mediated Gene Silencing
siRNA
siRNA triggers endonucleolytic
cleavage of perfectly-matched
complementary targets
Cleavage is catalyzed
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)
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)
Prevalence of and Regulation by miRNAs
At least 1400 miRNA-encoding genes in humans
miRNAs regulate ~50% of the human transcriptome
miRNAs fine tune the expression of proteins in a cell
Organismal Complexity May Be Due to Differences
in Regulation of Gene Expression
Number of protein-coding
genes are similar in animals
There is a continuous acquisition
of novel miRNAs during evolution
Lineage-specific loss of miRNAs also occurs
miRNA complexity correlates with an
increase in morphological complexity
There are now estimated to
be 1,424 miRNAs in humans
from Technau, Nature 455, 1184 (2008)
let-7 is a Heterochronic Gene in C. elegans
Mutations in heterochronic genes cause
temporal cell fate transformations that
are altered relative to the timing
of events in other cells or tissues
let-7 mutations cause an
overproliferation of seam cells
Overproliferation of cells is a
characteristic of stem cells and cancer
from Büssing et al., Trends Mol.Med. 14, 400 (2008)
Regulation of Differentiation by let-7
let-7 levels are reduced in stem cells
Lin28 promotes reprogramming
by inhibition of let-7 maturation
from Viswanathan and Daley, Cell 140, 445 (2010)
Reprogramming to iPS Cells
Oct4
Sox2
Klf4
c-Myc
or
Oct4
Sox2
NANOG
Lin28
Lin28 represses let-7
Is let-7 repression important for establishment of pleuripotent state?
c-Myc is a let-7 target, so Lin28 replaces c-Myc
Links of let-7/Lin28 to Cancer
let-7 is a tumor suppressor
The oncogenes c-Myc, K-Ras, and cyclin D1 are let-7 targets
Lin28 is an oncogene that is activated in 15% of human tumors
Lin28 is also a let-7 target
let-7
Lin28
double-negative feedback loop
Lin28 Prevents let-7 Maturation
let-7 promotes differentiation
pri-let-7 is processed to pre-let-7 by Drosha
After export, pre-let-7 is processed by Dicer
Lin28 recruits TUTase with uridylates the
miRNA which promotes let-7 degradation
During differentiation, let-7 targets
Lin28 mRNA, which reinforces
developmental commitment
from Heo et al., Mol.Cell 32, 276 (2008)
Summary of let-7/Lin28 Regulatory Pathways
Lin28 prevents let-7 muturation
let-7 promotes differentiation
and prevents transformation
Lin28 promotes reprogramming
or transformation
from Viswanathan and Daley, Cell 140, 445 (2010)
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)
A MicroRNA Regulates Neuronal Differentiation
by Controlling Alternative Splicing
miR-124 targets a component of a
repressor of neuron-specific genes
miR-124 results in reduced
expression of PTBP1 leading
to the accumulation of PTBP2
PTBP2 results in a global switch to neuronspecific alternative splicing patterns
from Makeyev et al., Mol.Cell 27, 435 (2007)
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)
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)
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)
Role of MicroRNAs and Epigenetics in Cancer
EZH2 overexpression promotes cell proliferation
Expression of EZH2 is inhibited by miR-101
miR-101 expression decreases during prostate cancer progression
from Varambally et al., Science 322, 1695 (2008)
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)
DNA Vector-based RNAi
from Shi, Trends Genet. 19, 9 (2003)
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)
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)
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)
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)
Inhibition of Endogenous miRNA function
miRNA sponges
Vectors express multiple
copies of miRNA target sites
Endogenous miRNA is
saturated and prevented from
silencing its natural product
from Brown and Naldini, Nature Rev.Genet. 10, 578 (2009)
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)
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
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)
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
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
Polyomaviruses also have
viral miRNA that targets TAg
Infection with Py mutant lacking
the miRNA resulted in no difference
in viral load or immune response
from Sarnow et al., Nature Rev.Microbiol. 4, 651 (2006)
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 Rev.Microbiol. 4, 651 (2006)
A MicroRNA was Thought to Protect 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
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 inhibits RNAi
miR-122 increases HCV
replication in the liver
from Sarnow et al., Nature Rev.Microbiol. 4, 651 (2006)
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
from Aravin et al., Science 318, 761 (2007)
piRNAs display 10 nt
complementarity at their 5’-ends
Model for Biogenesis of piRNAs that Target Mobile Elements
Pool of piRNAs bound 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)
from Büssing et al., Trends Mol.Med. 14, 400 (2008)