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

Examples of Homology-dependent Gene Silencing

TGS – Pairing of tightly linked homologous loci induces methylationTranscriptional Gene Silencing

PTGS – Transcript-specific degradationPost-transcriptional Gene Silencing

SAS – Spread of PTGSSystemic Acquired Silencing

RIP – Induction of C-T transitionsRepeat-induced Point Mutation

RNAiRNA 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 McManus and Sharp, Nature Rev.Genet. 3, 737 (2002)

Response of Mammalian Cells to Long dsRNA

Long dsRNA induces interferon response in vertebrates

PKR phosphorylates eIF2 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

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 Lodish et al., Molecular Cell Biology, 6th ed. Fig 21-6

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

miRNAs Regulate Development in C. elegans

from Li and Hannon, Nature Rev.Genet. 5, 522 (2004)

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

miRNAs have imperfect complementarity to their target mRNA and inhibit translation

siRNAs form perfect duplex with their target mRNA and trigger mRNA degradation

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

Triggers of RNAi-Mediated Gene Silencing in Mammals

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

from Chen and Rajewsky, Nature Rev.Genet. 8, 93 (2007)

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

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 Li and Hannon, Nature Rev.Genet. 5, 522 (2004)

Domain Structures of Dicer Enzymes

Dicer generates mature miRNA and siRNA in the cytoplasm

In Arabidopsis, DCL-1 contains NLS and processes pri-miRNA

Strand Selection of Processed siRNA into the RISC

from Sontheimer, Nature Rev.Mol.Cell Biol. 6, 127 (2005)

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

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)

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)

Overview of RNA-Mediated Gene Silencing

from Eulalio et al., Nature Rev.Mol.Cell Biol. 8, 9 (2007)

siRNA triggers endonucleolytic cleavage of perfectly-matched complementary targets

The resulting mRNA fragments are degraded

miRNA triggers accelerated deadenylation and decapping of partially-complementary targets and requires Argonaute proteins and a P-body component

Cleavage is catalyzed by Argonaute proteins

miRNA represses translation

siRNA

miRNA

Regulation of siRNA Levels in C. elegans

from Timmons, BioEssays 26, 715 (2004)

RNA-dependent RNA polymerase amplifies siRNA

RRF-3 prevents siRNA amplification

ERI-1 is an siRNA-specific RNase

At least 800 miRNA-encoding genes in humans

At least 5000 genes are regulated by miRNAs

Prevalence of and Regulation by miRNAs

from Technau, Nature 455, 1184 (2008)

miRNA complexity correlates with an increase in morphological complexity

Number of protein-coding genes are similar in animals

Organismal Complexity May Be Due to Differences in Regulation of Gene Expression

Human Accelerated Regions

from Pollard et al., Nature 443, 167 (2006)

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

HAR1-HAR49 are sequences that are highly conserved among mammals, but have exhibited rapid, recent sequence divergence

Most HARs occur outside protein coding regions

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.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

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

The undifferentiated state of malignant cells is correlated with a decrease in miRNA expression

from He et al., Nature 435, 828 (2005) Lu et al., Nature 435, 834 (2005)

Down-regulation of all miRNAs enhanced tumor growth

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

miRNAs Suppress Breast Cancer Metastasis

from Tavasoie et al., Nature 451, 147 (2008)

from Steeg, Nature 449, 671 (2007)

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

EZH2 overexpression promotes cell proliferation

Expression of EXH2 is inhibited by miR-101

miR-101 expression decreases during prostate cancer progression

Role of MicroRNAs and Epigenetics in Cancer

from Varambally et al., Science 322, 1695 (2008)

Immunostimulatory Effects of dsRNA

from Kim and Rossi, Nature Rev.Genet. 8, 173 (2007)

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

DNA Vector-based RNAi

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

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

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 Dykxhoorn and Lieberman, Cell 126, 231 (2006)

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 Rossi et al., Nature Biotechnol. 23, 682 (2005)

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

Cell-Specific Delivery of siRNA

Non-specific siRNA Inhibition of Angiogenesis

from Kalluri and Kanasaki, Nature 452, 543 (2008)

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

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

Viral suppressors of RNA silencing (VSR) inhibit the RNAi pathway

Host cell-encoded miRNAs inhibit or facilitate viral replication

from Sarnow et al., Nature Reviews Microbiol. 4, 651 (2006)

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

Function of SV40 miRNA

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

LAT is the only viral gene expressed during latent infection in neurons

miR-LAT is generated from the LAT gene

A MicroRNA Protects HSV-1-infected Neurons from Apoptosis

miR-LAT downregulates TGF- 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 Sarnow et al., Nature Reviews Microbiol. 4, 651 (2006)

Cellular miRNAs Modulates Viral Infection

Tas is a PFV-1-encoded protein that inhibits RNAi

miR-32 inhibits viral replication

PFV-1 replication is stimulated by a plant VSR implicating the role of small RNAs in the viral life cycle

miR-122 increases HCV replication in the liver

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’-endsfrom Aravin et al., Science 318, 761 (2007)

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)