Homology-dependent Gene Silencing – The World in 1999
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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.

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

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From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

Small RNAs

from tenOever, Nature Rev.Microbiol. 11, 169 (2013)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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)


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)


From wu and morris curr opin genet dev 9 237 1999

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


From wu and morris curr opin genet dev 9 237 1999

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


From wu and morris curr opin genet dev 9 237 1999

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 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)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

Triggers of RNAi-Mediated Gene Silencing in Mammals

from Mittal, Nature Rev.Genet. 5, 355 (2004)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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, BioEssays26, 715 (2004)


From wu and morris curr opin genet dev 9 237 1999

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


From wu and morris curr opin genet dev 9 237 1999

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, Nature455, 1184 (2008)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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, Cell140, 445 (2010)


From wu and morris curr opin genet dev 9 237 1999

Reprogramming to iPS Cells

Oct4

Sox2

Klf4

c-Myc

Oct4

Sox2

NANOG

Lin28

or

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


From wu and morris curr opin genet dev 9 237 1999

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


From wu and morris curr opin genet dev 9 237 1999

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 Drosha-

mediated 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)


From wu and morris curr opin genet dev 9 237 1999

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


From wu and morris curr opin genet dev 9 237 1999

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 neuron-

specific alternative splicing patterns

from Makeyev et al., Mol.Cell27, 435 (2007)


From wu and morris curr opin genet dev 9 237 1999

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., Nature435, 828 (2005)

Lu et al., Nature435, 834 (2005)

Lujambio and Lowe, Nature482, 347 (2012)


From wu and morris curr opin genet dev 9 237 1999

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., Nature451, 147 (2008)


From wu and morris curr opin genet dev 9 237 1999

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, Nature482, 347 (2012)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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


From wu and morris curr opin genet dev 9 237 1999

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

PTENP1 RNA is a ceRNA that

enhances PTEN expression by

competing for a shared miRNA

from Rigoutsos, Nature465, 1016 (2010)


From wu and morris curr opin genet dev 9 237 1999

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., Nature505, 344 (2014)


From wu and morris curr opin genet dev 9 237 1999

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, Science340, 440 (2013)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

DNA Vector-based RNAi

from Shi, Trends Genet.19, 9 (2003)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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, Cell126, 231 (2006)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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, Science297, 1818 (2002)


From wu and morris curr opin genet dev 9 237 1999

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


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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

Paper retracted – 2008. Repeatedly

unable to detect miRNA

from Gupta et al., Nature 442, 82 (2006)


From wu and morris curr opin genet dev 9 237 1999

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


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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)


From wu and morris curr opin genet dev 9 237 1999

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


From wu and morris curr opin genet dev 9 237 1999

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., Science318, 761 (2007)


From wu and morris curr opin genet dev 9 237 1999

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., Science318, 761 (2007)


From wu and morris curr opin genet dev 9 237 1999

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, Nature509, 570 (2014)

Masc promotes male-specific

splicing of doublesex


From wu and morris curr opin genet dev 9 237 1999

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


From wu and morris curr opin genet dev 9 237 1999

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., Nature482, 331 (2012)

CRISPR is a bacterial

memory of past invasions


From wu and morris curr opin genet dev 9 237 1999

CRISPR RNA Biogenesis and Interference

CRISPR loci are transcribed

and processed into crRNAs

CRISPR RNA is processed by CRISPR-

specific 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., Nature482, 331 (2012)


From wu and morris curr opin genet dev 9 237 1999

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, Science344, 707 (2014)

Self targeting is avoided since

the CRISPR locus lacks PAMs


From wu and morris curr opin genet dev 9 237 1999

The CRISPR-Cas9 System is Used for Targeted Genome Editing

from Charpentier and Doudna, Nature495, 50 (2013)


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