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

Small RNAs

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

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 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)

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

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)

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

from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011)

The cap structure is recognized by eIF4F

Poly(A) is recognized by PABPC

PABPC interacts with eIF4G

Recruitment of the preinitiation complex is increased

miRNAs Promote mRNA Deadenylation

from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011)

miRNA guide strand associates with AGO

AGO interacts with GW182

GW182 may compete with eIF4G for binding to PABPC and prevents mRNA circularization

Assembly of AGO-GW182-PABPC complex triggers deadenylation by CAF1-CCR4-NOT

GW182 may reduce the affinity of PABPC for the poly(A) tail

Fate of Deadenylated mRNAs

from Huntzinger and Izaurralde, Nature Rev.Genet. 12, 99 (2011)

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

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

Secretion of miRNAs

from Chen et al., Trends Cell Biol. 22, 125 (2012)

Specific miRNAs can be preferentially sorted into vesicles and delivered to recipient cells

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 1400 miRNA-encoding genes in humans

miRNAs regulate ~50% of the human transcriptome

Prevalence of and Regulation by miRNAs

miRNAs fine tune the expression of proteins in a cell

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

There is a continuous acquisition of novel miRNAs during evolution

Lineage-specific loss of miRNAs also occurs

There are now estimated to be 1,424 miRNAs in humans

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

let-7 is a Heterochronic Gene in C. elegans

from Büssing et al., Trends Mol.Med. 14, 400 (2008)

from Viswanathan and Daley, Cell 140, 445 (2010)

Regulation of Differentiation by let-7

let-7 levels are reduced in stem cells

Lin28 promotes reprogramming by inhibition of let-7 maturation

Reprogramming to iPS Cells

Oct4Sox2Klf4c-Myc

Oct4Sox2NANOGLin28

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

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

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)

Lin28 Prevents let-7 Maturation

let-7 promotes differentiation

from Viswanathan and Daley, Cell 140, 445 (2010)

Summary of let-7/Lin28 Regulatory Pathways

Lin28 prevents let-7 muturation

let-7 promotes differentiation and prevents transformation

Lin28 promotes reprogramming or transformation

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 mRNA expression data

miRNA expression is lower in cancers than in most normal tissues, but expression of some miRNAs is increased

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) Lujambio and Lowe, Nature 482, 347 (2012)

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 and Breast Cancer Metastasis

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

miR-10b and miR-9 induce metastasis

EZH2 (a PcG protein) overexpression promotes cell proliferation

Expression of EZH2 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)

miR-29 inhibits DNMT3A and DNMT3B in lung cancer

from Lujambio and Lowe, Nature 482, 347 (2012)

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 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

Inhibition of Endogenous miRNA function

Vectors express multiple copies of miRNA target sites

miRNA sponges

Endogenous miRNA is saturated and prevented from silencing its natural product

from Brown and Naldini, Nature Rev.Genet. 10, 578 (2009)

Pseudogene transcripts can act as miRNA sponges

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 Rev.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

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 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 was Thought to Protect 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 Rev.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

miR-122 stabilizes the HCV genome by binding the 5’-UTR

miR-122 Protects the HCV Genome From Degradation

from Garcia-Sastre and Evans, Proc.Nat.Acad.Sci. 110, 1571 (2013)

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

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

miRNA Encoded by an RNA Virus

from Cullen, Proc.Nat.Acad.Sci. 109, 2695 (2012)

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)

Large ncRNAs

Much of the genome is transcribed

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 Wiedenheft et al., Nature 482, 331 (2012)

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

CRISPR is a bacterial memory of past invasions

cas genes are linked to the CRISPR locus and are involved in integration, processing and interference

CRISPR RNA Biogenesis and Interference

from Wiedenheft et al., Nature 482, 331 (2012)

CRISPR loci are transcribed and processed into crRNAs

CRISPR RNA is processed by CRISPR-specific endonucleases or by RNaseIII cleavage of a tracrRNA-DNA duplex

crRNAs associated with Cas proteins, recognize and cleave foreign nucleic acids