Transcript Posttranscriptional Gene Silencing-2015

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)
miRNA Biogenesis
pri-miRNA is cleaved by Drosha
at the base of a stem-loop structure
pre-miRNA is exported from the nucleus
by exportin 5 and cleaved by Dicer
The duplex is loaded onto Argonaute protein
miRNA* is expelled to produce the RISC
from Ameres and Zamore, Nature Rev.Mol.Cell Biol. 14, 475 (2013)
miRNA Target Recognition in Plants
from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011)
The 3’-end of plant miRNAs usually are modified with a 2’O-methyl group
Plant miRNAs usually recognize fully complementary binding sites located in the ORF
The PIWI domain of AGO cleaves the mRNA target between nucleotides 10-11 opposite the miRNA
miRNA Target Recognition in Animals
from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011)
Animal miRNAs usually recognize partially complementary sites located in the 3’-UTR
Complementarity within the seed region is the major determining of binding to the target
Regulation of Small RNA Levels
The presence of highly complementary
target RNA triggers tailing and trimming
In flies, small RNAs can be stabilized
by methylation at the 3’-end
from Ameres and Zamore, Nature Rev.Mol.Cell Biol. 14, 475 (2013)
Triggers of RNAi-Mediated Gene Silencing in Mammals
from Mittal, Nature Rev.Genet. 5, 355 (2004)
Argonaute Proteins
Argonaute proteins bind small RNAs and identify RNA targets by base-pairing
Target silencing can occur by
Target mRNA degradation
Translation inhibition
Recruitment of chromatin-modifying activities
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)
Mechanisms of miRNA Sequence Diversification
Differential processing results in
miRNAs with different seed sequences
In cells containing adenosine
deaminase, A is converted to I
from Ameres and Zamore, Nature Mol.Cell Biol. 14, 475 (2013)
Causes of Off-Target Effects
Sequence-based homology with non-target transcripts
Aberrant processing or RNA editing
General cell pertubations due to large amounts of RNA
Incorporation of the passenger strand into the RISC
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)
Mechanism of miRNA Action
miRNAs Induce mRNA Degradation and Inhibit Translation
miRNA-AGO binds to partially
complementary sites in the target mRNA
AGO recruits GW182 proteins
from Izaurralde, Science 349, 380 (2015)
miRNA-mediated Inhibition of Target mRNA Translation
Translation repression accounts
for a small part of target repression
GW182 recruits deadenylase complexes
to initiate mRNA degradation
The miRISC interferes with the helicase
function of eIF4A to inhibit translation initiation
from Izaurralde, Science 349, 380 (2015)
miRNA-mediated mRNA Degradation
mRNA degradation is the dominant
effect of miRNA-mediated regulation
The decapping complex is
recruited to the miRISC
mRNA decapping makes the 5’-end
accessible to the XRN1 nuclease
from Izaurralde, Science 349, 380 (2015)
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
from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011)
Recruitment of the preinitiation
complex is increased
miRNAs Effects on Preinitiation Complex Formation
miRISC-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
Preinitiation complex recruitment is inhibited
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
from Eulalio et al., Nature Rev.Mol.Cell Biol. 8, 9 (2007)
miRNA degrades mRNA
and represses translation
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)
Tumor-derived Exosomes Initiate Tumor Growth in Normal Cells
OncomiRs are incorporated into exosomes
which are taken up by normal cells
Exosomes may be used as
biomarkers for cancer diagnosis
Exosome mimetics containing miRNA sponges
could fuse with normal cells and tumorderived exosomes to neutralize oncomiRs
from Anastasiadou and Slack, Science 346, 1459 (2014)
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
The human genome has the potential to encode >1500 miRNAs
miRNAs control the expression of >50% of human proteins
mRNAs can undergo negative selection to avoid a seed match
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)
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)
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
from Dykxhoorn and Lieberman, Cell 126, 231 (2006)
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
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)
siRNA-mediated Pericentric Heterochromatin Formation in S. pombe
Bidirectional transcription produces
dsRNA that is processed into siRNA
RNA-dependent RNA polymerase
produces additional dsRNA and
more siRNA is generated
siRNA loaded onto Ago1 is
guided to the nascent transcript
from Castel and Martienssen, Nature Rev.Genet. 14, 100 (2013)
Clr4 is recruited which methylates H3K9
3’-End Processing Prevents Transcriptional Silencing in S. pombe
The effect of RNAi on
chromatin is inconsistent
Paf1C is essential for 3’-end
processing of mRNA
Paf1C prevents siRNA-mediated
heterochromatin formation
from Zaratiegui, Nature 520, 162 (2015)
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)
PIWI and piRNA
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
Overview of piRNA Biogenesis in the Drosophila Female Germ Line
Precursor piRNAs are transcribed
from piRNA clusters
Processing generates antisense
piRNA containing a 5’-U
piRNA is loaded onto AUB or PIWI
and cleaves transposon RNA to
produce sense piRNA containing 10A
Additional antisense piRNA is produced by
AGO3-mediated cleavage of piRNA precursors
from Castel and Martienssen, Nature Rev.Genet. 14, 100 (2013)
Precursor piRNA Production in Drosophila
How are piRNA transcripts
selectively processed into piRNAs?
Rhino is an HP1 homolog that interacts with
chromatin, UAP56, and precursor piRNAs
UAP56 and Vasa are RNA helicases
that facilitate transport of precursor
piRNAs to their site of processing
from Luteijn and Ketting, Nature Rev.Genet. 14, 523 (2013)
Primary piRNA Biogenesis in Drosophila
ssRNA are precursors to piRNAs and
production of piRNAs are Dicer-independent
Zucchini may be the nuclease
that cleaves piRNA precursors
Piwi proteins may selectively bind
RNAs that contain a 5’-U
The 3’-end is trimmed and methylated to
protect the piRNA from uridylation and degradation
from Luteijn and Ketting, Nature Rev.Genet. 14, 523 (2013)
Secondary piRNA Biogenesis in Drosophila
piRNA clusters often contain transposons
Secondary piRNA processing ensures that the
piRNA pool relates to the expression of their targets
Aubergine bound to the primary piRNA cleaves
the target RNA which is taken up by Ago3
The 3’-end is trimmed and methylated and binds
to a precursor piRNA which is cleaved by Ago3
from Luteijn and Ketting, Nature Rev.Genet. 14, 523 (2013)
Role of piRNA in the Nucleus
Piwi-piRNA can enter the nucleus and is
responsible for H3K9 methylation to
spread heterochromatin at transposon loci
Transposon RNA is down-regulated
from Luteijn and Ketting, Nature Rev.Genet. 14, 523 (2013)
Role of piRNA in Sex Determination in Silkmoths
from Watanabe and Lin, Mol.Cell 56, 18 (2014)
WZ – female
ZZ - male
Sexual development is controlled by the sex-specific splicing of doublesex mRNA
Masc promotes male-specific splicing of doublesex
piRNAs are transcribed from W chromosome in females and reduces Masc mRNA levels
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 Type II CRISPR System is Used for Targeted Genome Editing
sgRNA generates a predictable
ds break adjacent to a PAM site
Break is repaired by
NHEJ to knockout gene
Presence of a homologous template
allows genome editing using HR
from Barrangou and Marraffini, Mol.Cell 54, 234 (2014)
sgRNA-Guided Transcription Repression
A catalytically dead Cas9 and sgRNA
is targeted to any genomic location
Targeting to a promoter
represses transcription initiation
from Barrangou and Marraffini, Mol.Cell 54, 234 (2014)
Targeting to the template strand
represses transcription elongation
dCas9 Targeting of Enzymatic Activities to Specific Genomic Locations
dCas9 can be fused
to any functional domain
Chimeric protein can be targeted
to any genomic location by sgRNA
from Barrangou and Marraffini, Mol.Cell 54, 234 (2014)
Avoiding Off-target Effects in Genome Editing
Cas9 is converted to a nickase
Two sgRNAs directs nicks on
opposite strands at adjacent sites
from Ran et al., Cell 154, 1380 (2013)
CRISPR-Cas9 Targeting of ssRNA
from O’Connell et al., Nature 516, 263 (2014)
A PAMmer anneals upstream of the target sequence
A 5’-extended PAMmer is required for CRISPR-CAS9 cleavage of ssRNA