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Homology-dependent Gene Silencing – The World in 1999
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)
Small RNAs
from tenOever, Nature Rev.Microbiol. 11, 169 (2013)
Response to Virus Infection in Chordates
Viral dsRNA is recognized by PRRs
in the cytoplasm or TLRs in endosomes
Induce expression of type I interferons
Leads to transactivation of >250 genes
Slows viral infection and allows
time for an adaptive immune response
from tenOever, Nature Rev.Microbiol. 11, 169 (2013)
viRNAs are an Antiviral Innate Immune System
viRNAs are derived from the
virus and loaded onto the RISC
viRNAs bind the viral RNA
target with perfect complementarity
and eliminates the target
Chordates do not produce viRNA
from tenOever, Nature Rev.Microbiol. 11, 169 (2013)
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 the precursor to a 22 ntlong 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)
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)
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 that results from variations in
Drosha or Dicer processing generates isomiRs
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)
Secretion of miRNAs
Specific miRNAs can be
preferentially sorted into vesicles
and delivered to recipient cells
from Chen et al., Trends Cell Biol. 22, 125 (2012)
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
Transfection of ESCC (ES cell-specific cell cycle-regulating)
miRNAs can generate ES cells without protein-encoding factors
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
Lin28a and Lin28b repress let-7
biogenesis by two distinct mechanisms
Lin28a recruits TUTase which uridylates the
miRNA and promotes let-7 degradation
Lin28b inhibits Droshamediated processing of let-7
During differentiation, let-7 targets
Lin28 mRNA, which reinforces
developmental commitment
from Thornton and Gregory, Trends Cell Biol. 22, 474 (2012)
Summary of Lin28 let-7 Regulation of Differentiation and Oncogenesis
from Thornton and Gregory, Trends Cell Biol. 22, 474 (2012)
Lin28 prevents let-7 muturation
let-7 promotes differentiation and prevents transformation
Lin28 promotes reprogramming or transformation
ESCC miRNAs maintain Lin28 expression
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 mRNA expression data
miRNA expression is lower in cancers than in most normal tissues,
but expression of some miRNAs is increased
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)
Lujambio and Lowe, Nature 482, 347 (2012)
miRNAs and 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
miR-10b and miR-9 induce metastasis
from Tavasoie et al., Nature 451, 147 (2008)
Role of MicroRNAs and Epigenetics in Cancer
EZH2 (a PcG protein) 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)
miR-29 inhibits DNMT3A and DNMT3B in lung cancer
from Lujambio and Lowe, Nature 482, 347 (2012)
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
Pseudogene transcripts can
act as miRNA sponges
from Brown and Naldini, Nature Rev.Genet. 10, 578 (2009)
Competitive Endogenous RNAs (ceRNAs)
70-90% of the human genome is transcribed, but less
than 2% of the genome encodes protein-coding genes
The human transcriptome contains 21.000 protein-coding genes,
9,000 small RNAs, 10,000-32,000 lncRNAs and 11,000 pseudogenes
All RNA transcripts that contain miRNA binding sites
that regulate each other by competing for shared miRNAs
ceRNAs can fine-tune gene expression
Regulation of PTEN Levels by a Pseuodogene
The expression level of PTEN is crucial
for its tumor suppressive function
PTEN expression is
downregulated by miRNAs
PTENP1 is a pseudogene which
contains the same MRE in the 3’-UTR
from Rigoutsos, Nature 465, 1016 (2010)
PTENP1 RNA is a ceRNA that
enhances PTEN expression by
competing for a shared miRNA
The PTEN ceRNA Network
PTEN expression levels are
regulated by a large network of
miRNAs, mRNAs, and ceRNAs
The PTEN ceRNA interactions are part
of a regulatory layer comprising of more
than 248,000 miRNA-mediated interactions
from Tay et al., Nature 505, 344 (2014)
Circular RNAs can be microRNA Sponges
Human fibroblasts have 25,000 circRNAs
derived from 15% of transcribed genes
The splicing machinery is
involved in circRNA biogenesis
circRNAs are resistant to
degradation triggered by miRNAs
from Wilusz and Sharp, Science 340, 440 (2013)
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)
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)
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)
miR-122 stabilizes the HCV
genome by binding the 5’-UTR
miR-122 Protects the HCV Genome From Degradation
Xrn1 is a cytoplasmic exonuclease
that normally degrades HCV RNA
miR-122 increases HCV RNA stability
by shielding the genome against Xrn1
miR-122 also enhances HCV
RNA replication that is independent
on its action against Xrn1
from Garcia-Sastre and Evans, Proc.Nat.Acad.Sci. 110, 1571 (2013)
miRNA Encoded by an RNA Virus
Most miRNAs are transcribed by pol II
and processed by Drosha in the nucleus
MHV68 pri-miRNA is transcribed
by pol III and processed by tRNase Z
BLV miRNA is transcribed by pol III
from Cullen, Proc.Nat.Acad.Sci. 109, 2695 (2012)
The Drosophila PIWI phenotype –
P-element-induced wimpy testis
PIWIs and piRNAs are enriched in the germline
PIWI mutations result in infertility
piRNA-PIWI pathway is
involved in transposon silencing
PIWI depletion results in an upregulation
of transposon mRNA expression
PIWIs are expressed in some somatic
cells and is important for stem cell
function and regeneration in planarians
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)
Role of piRNA in Sex Determination in Silkmoths
WZ – female
ZZ - male
Sexual development is controlled by the
sex-specific splicing of doublesex mRNA
piRNAs are transcribed from W chromosome
in females and reduces Masc mRNA levels
from Marek, Nature 509, 570 (2014)
Masc promotes male-specific
splicing of doublesex
Large ncRNAs
Much of the genome is transcribed
Human genome encodes
21,000 protein-coding genes
9,000 small RNAs
10,000 – 32,000 lncRNAs
11,000 pseudogenes
Many large ncRNAs contain modular domains that interact with chromatin regulators
Large ncRNAs can function as a molecular scaffold that forms a unique functional complex
CRISPR is a Bacterial Defense Based on Small RNA
CRISPR contains repeats separated by
unique spacers that arise from integration
of short fragments of foreign DNA
cas genes are linked to the CRISPR
locus and are involved in integration,
processing and interference
from Wiedenheft et al., Nature 482, 331 (2012)
CRISPR is a bacterial
memory of past invasions
CRISPR RNA Biogenesis and Interference
CRISPR loci are transcribed
and processed into crRNAs
CRISPR RNA is processed by CRISPRspecific endonucleases or by RNaseIII
cleavage of a tracrRNA-RNA duplex
crRNAs associated with Cas proteins,
recognize and cleave foreign nucleic acids
from Wiedenheft et al., Nature 482, 331 (2012)
Cas9 Targeting and ds Break Formation
Cas9 + crRNA + tracrRNA or
(sgRNA) binds to PAM sites
Recognition of PAM promotes
local unwinding and interrogates
flanking DNA for the target
PAM binding activates the Cas9-RNA
nuclease activity and generates a ds break
Specificity is determined by the crRNA sequence
Cas9 remains bound after cleavage to
allow recruitment of DNA repair machinery
from Barrangou, Science 344, 707 (2014)
Self targeting is avoided since
the CRISPR locus lacks PAMs
The CRISPR-Cas9 System is Used for Targeted Genome Editing
from Charpentier and Doudna, Nature 495, 50 (2013)