RNAi: Insight, Mechanisms RNAi: Insight, Mechanisms and Potentialand Potential

Nobel Prize in Physiology or Nobel Prize in Physiology or Medicine awarded toMedicine awarded toA. Fire and C. MelloA. Fire and C. Mello

What is RNA interference (RNAi)?What is RNA interference (RNAi)?““The Process by which dsRNA silences The Process by which dsRNA silences gene expression...”gene expression...”

Degradation of mRNA or translation Degradation of mRNA or translation inhibitioninhibition

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What are sense and antisense What are sense and antisense RNA?RNA?

Messenger Messenger RNA (mRNA) RNA (mRNA) is single-is single-stranded, stranded, called "sense" called "sense" because it because it results in a results in a gene product gene product (protein). (protein).

5´   C U U C A  3´     mRNA3´   G A A G U  5´     Antisense RNA

What are sense and antisense What are sense and antisense RNA?RNA?

Antisense Antisense molecules molecules interact with interact with complementary complementary strands of strands of nucleic acids, nucleic acids, modifying modifying expression of expression of genes.genes.

5´   C U U C A  3´     mRNA3´   G A A G U  5´     Antisense RNA

RNAi termsRNAi terms

dsRNA: double stranded RNA, longer than dsRNA: double stranded RNA, longer than 30 nt30 nt

miRNA: microRNA, 21-25 nt. miRNA: microRNA, 21-25 nt. – Encoded by endogenous genesEncoded by endogenous genes

siRNA: ssiRNA: smallmall-interfering RNA, 21-25 nt.-interfering RNA, 21-25 nt.– Mostly exogenous originMostly exogenous origin

Nature, it seems, is the popular gameNature, it seems, is the popular game

for milliards, and milliards, and milliardsfor milliards, and milliards, and milliards

of particles playing their infinite gameof particles playing their infinite game

of billiards and billiards and billiardsof billiards and billiards and billiards

Piet Hein (1966) *Piet Hein (1966) *

Piet Hein (December 16, 1905 - April 17, 1996), a Danish scientist, mathematician, inventor, author, and poet; known for his short poems in the form of grook. A grook ("gruk“, short for "GRin & sUK" ("laugh & sigh" in Danish) is a form of short aphoristic poem.

*From ‘Critical Mass: How One Thing Leads To Another’ by Phillip Ball *From ‘Critical Mass: How One Thing Leads To Another’ by Phillip Ball

RNAi like phenomenaRNAi like phenomena

PlantsPlants– PetuniasPetunias

FungiFungi– NeurosporaNeurospora

AnimalsAnimals– Caenorhabditis elegansCaenorhabditis elegans

Alternate terms to RNAiAlternate terms to RNAi

PTGS (Posttranscriptional Gene PTGS (Posttranscriptional Gene Silencing)Silencing)

CosuppressionCosuppression

QuellingQuelling

Virus-induced gene silencingVirus-induced gene silencing

1990-Petunias1990-Petunias

Napoli et al. defined an RNAi-like Napoli et al. defined an RNAi-like phenomenon and called it phenomenon and called it ““cosupressioncosupression.”.”

chalcone synthase (CHS), a key enzyme chalcone synthase (CHS), a key enzyme in flavonoid biosynthesis, the rate-limiting in flavonoid biosynthesis, the rate-limiting enzyme in anthocyanin biosynthesis, enzyme in anthocyanin biosynthesis, responsible for the purple coloration.responsible for the purple coloration.

Overexpression of chalcone Overexpression of chalcone synthase in petunias unexpectedly synthase in petunias unexpectedly

resulted in white petuniasresulted in white petunias

The levels of endogenous as The levels of endogenous as well as introduced CHS were well as introduced CHS were 50-fold lower than in wild-50-fold lower than in wild-type petunias, which led the type petunias, which led the authors to hypothesize that authors to hypothesize that the introduced transgene the introduced transgene was “was “cosuppressingcosuppressing” the ” the endogenous CHS gene.endogenous CHS gene.

http://www.scq.ubc.ca/?p=265

1992-The mold1992-The mold

Carlo Cogoni and Guiseppe Macino of the Carlo Cogoni and Guiseppe Macino of the Università di Roma La Sapienza in Italy Università di Roma La Sapienza in Italy introduced a gene needed for introduced a gene needed for carotenoid carotenoid synthesis in the mold synthesis in the mold Neurospora crassa:Neurospora crassa:– The introduced gene The introduced gene led to inactivation of the led to inactivation of the

mold's own genemold's own gene in about 30% of the in about 30% of the transformed cells. They called this gene transformed cells. They called this gene inactivation "inactivation "quellingquelling."."

A rosette of the asci

1995-The worm1995-The worm

Guo and Kemphues studied Guo and Kemphues studied par-1par-1 gene gene during embryogenesisduring embryogenesis

The worm, The worm, C. elegansC. elegans– has a fixed lineage:has a fixed lineage: hypodermishypodermis, , intestineintestine, ,

gonadsgonads– asymmetric divisionsasymmetric divisions

1995- The worm1995- The worm

Guo and Kemphues Guo and Kemphues first studied Par-1 first studied Par-1 gene mutantsgene mutants– Division: Division:

AsymmetricAsymmetricsymmetricsymmetric– P-granule distribution P-granule distribution

Guo and Kemphues, 1995Guo and Kemphues, 1995

Both the antisense and sense Both the antisense and sense strands effectively silencedstrands effectively silenced

wildtype

Par-1 RNAi

The reasonings about the wonderful and The reasonings about the wonderful and intricate operations of Nature are so full of intricate operations of Nature are so full of uncertainty, that, as the Wise-man truly uncertainty, that, as the Wise-man truly observes, hardly do we guess aright at the observes, hardly do we guess aright at the things that are upon earth, and with labour things that are upon earth, and with labour do we find the things that are before us.do we find the things that are before us.

Stephen Hales (1727)Stephen Hales (1727)Stephen Hales (September 17, 1677 - January 4, 1761) was an English physiologist, chemist and inventor; studied the role of air and water in the maintenance of both plant and animal life.

‘‘Antisense’ Technology?Antisense’ Technology?

Sense RNA silences yet no hybridization of Sense RNA silences yet no hybridization of sense RNA with sense mRNA is expected!sense RNA with sense mRNA is expected!

Intronic and promoter sequences do not silence.Intronic and promoter sequences do not silence.

ssDNA or dsDNA does not work!ssDNA or dsDNA does not work!

Craig Mello at the Worm Meeting in Madison, Craig Mello at the Worm Meeting in Madison, Wisconsin coined the term Wisconsin coined the term ‘RNAi’‘RNAi’ and said that: and said that:– “ “ We can’t call it ‘antisense’ when ‘sense’ works as We can’t call it ‘antisense’ when ‘sense’ works as

well”*well”*

*Montgomery (2006) RNA interference: unraveling a mystery

Craig MelloCraig Mello

In 1996, C. Mello and his student S. In 1996, C. Mello and his student S. Driver also reported that sense RNAs Driver also reported that sense RNAs mimic antisense phenotype.mimic antisense phenotype.– Injection is made into a single site yet acts Injection is made into a single site yet acts

more systemically.more systemically.

Andrew FireAndrew Fire

In 1991, A. Fire successfully targeted In 1991, A. Fire successfully targeted genes by antisense constructs from genes by antisense constructs from transgenes. transgenes.

Sense constructs also exhibited silencing Sense constructs also exhibited silencing activity.activity.

Fire and MelloFire and Mello

Their paths crossed:Their paths crossed:– Mutual interest on a gene called pie-1.Mutual interest on a gene called pie-1.– Discuss what the sense and antisense Discuss what the sense and antisense

preparations have in commonpreparations have in common

Fire and MelloFire and Mello

did simple math…did simple math…

Sense + (-sense) Sense + (-sense) 0 (interference) 0 (interference)

Fire & Mello hypothesize on how Fire & Mello hypothesize on how sense works?sense works?

ssRNA ssRNA in vitroin vitro synthesized by bacteriophage synthesized by bacteriophage RNA pol might be contaminated by dsRNARNA pol might be contaminated by dsRNA

Bacteriophage RNA polymerase produce random Bacteriophage RNA polymerase produce random ectopic transcriptsectopic transcripts

DNA transgene arrays produce aberrant DNA transgene arrays produce aberrant RNA some with dsRNA characterRNA some with dsRNA character

certain repeated DNA sequences might be certain repeated DNA sequences might be expected to produce aberrant RNAs that would expected to produce aberrant RNAs that would then be capable of producing a silencing responsethen be capable of producing a silencing response

Fire & Mello hypothesize on how Fire & Mello hypothesize on how sense works?sense works?

1998-Fire et al. and Mello, Nature1998-Fire et al. and Mello, Nature

1998-Fire et al and Mello1998-Fire et al and Mello

Gel-purifiedGel-purified ssRNA ssRNA

Used purified ssRNA (Used purified ssRNA (antisense and antisense and sensesense) separately and also together.) separately and also together.

Tested ssRNA against Tested ssRNA against different genesdifferent genes for for specificityspecificity

Tested whether a Tested whether a general post-general post-transcriptional silencingtranscriptional silencing is in place. is in place.

Unc-22 (Uncoordinated 22)Unc-22 (Uncoordinated 22)

Codes for a non essential myofilamentCodes for a non essential myofilament

It is present several thousand copies/cellIt is present several thousand copies/cell

[If] There is some precision, there is some [If] There is some precision, there is some science.science.

Herbert Spencer (1880)Herbert Spencer (1880)

Herbert Spencer (27 April 1820 – 8 December 1903) was an English philosopher and prominent classic-liberal political theorist. Spencer analyzed human societies as evolving systems, and coined the term "survival of the fittest."

Injection for RNAiInjection for RNAi

6-10 adult hermaphrodites were injected 6-10 adult hermaphrodites were injected with 0.5x10with 0.5x1066-1x10-1x1066 molecules into each molecules into each gonadal arm.gonadal arm.

Unc-22 phenotypeUnc-22 phenotype

4-6 hours after injection, eggs 4-6 hours after injection, eggs collected.collected.

Screened for phenotypic Screened for phenotypic changeschanges– twichingtwiching

ExonExon SizeSize RNARNA PhenotypePhenotype

Exon 21-22Exon 21-22 742742 SenseSense

AntisenseAntisense

Sense+antisenseSense+antisense

WildtypeWildtype

WildtypeWildtype

Twicher (100%)Twicher (100%)

Exon 27Exon 27 10331033 SenseSense

AntisenseAntisense

Sense+antisenseSense+antisense

WildtypeWildtype

WildtypeWildtype

Twicher (100%)Twicher (100%)

Mex-3Mex-3mex-3 encodes two RNA binding proteins; mex-3 encodes two RNA binding proteins; in the early embryo, maternally provided in the early embryo, maternally provided Mex-3 is required for specifying the Mex-3 is required for specifying the identities of the anterior AB blastomere and identities of the anterior AB blastomere and its descendants, as well as for the identity its descendants, as well as for the identity of the P3 blastomere and proper of the P3 blastomere and proper segregation of the germline P granulessegregation of the germline P granules

Mex-3 RNAiMex-3 RNAi

b, Embryo from uninjected parent (showing normal pattern of endogenous mex-3 RNA20).

c, Embryo from a parent injected with purified mex-3B antisense RNA. Retain the mex-3 mRNA, although levels may be somewhat less than wild type.

d, Embryo from a parent injected with dsRNA corresponding to mex-3B; no mex-3 RNA is detected.

RNAi concentration and dose RNAi concentration and dose responseresponse

3.6x103.6x1066 molecules/gonad molecules/gonad– Sense phenocopied Sense phenocopied 1%1% of progeny of progeny– Antisense phenocopied Antisense phenocopied 11%11% of progeny of progeny– dsRNA phenocopies dsRNA phenocopies 100%100% progeny and at progeny and at

even 3x10even 3x1088 molecules/gonad. molecules/gonad.

Quantitative AssaysQuantitative Assays

Other possibilitiesOther possibilities

Sense+antisense in low saltSense+antisense in low salt

Rapid sequential injection of sense & Rapid sequential injection of sense & antisenseantisense– Both cause interferenceBoth cause interference– 1 hour apart injection of sense and antisense 1 hour apart injection of sense and antisense

leads to reduction in interference.leads to reduction in interference.

ConclusionsConclusions

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ConclusionsConclusions

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Nobody should be rich but those who Nobody should be rich but those who understand itunderstand it

Wolfgang von GoetheWolfgang von Goethe

Johann Wolfgang Goethe (28 August 1749 – 22 March 1832), a German polymath: a poet, novelist, dramatist, humanist, scientist, theorist, painter; the author of Faust and Theory of Colours

Nobel Prize in Physiology or Nobel Prize in Physiology or Medicine 2006Medicine 2006

Andrew FireAndrew Fire

Craig MelloCraig Mello

The LaureatesThe Laureates

Essense of Nobel PrizeEssense of Nobel Prize

Mello’s words after he had heard the news: “I seem too Mello’s words after he had heard the news: “I seem too young, and isn’t the gap unusually short?”young, and isn’t the gap unusually short?”

Most Nobel prizes are given many years after the Most Nobel prizes are given many years after the relevant discovery. The Fire and Mello award, given just relevant discovery. The Fire and Mello award, given just eight years after publication of their paper, is reminiscent eight years after publication of their paper, is reminiscent of Kary Mullis’s 1993 chemistry Nobel. That prize was of Kary Mullis’s 1993 chemistry Nobel. That prize was awarded for his 1985 invention of the polymerase chain awarded for his 1985 invention of the polymerase chain reaction — a method of gene amplification that invaded reaction — a method of gene amplification that invaded research labs just as fast and comprehensively as the research labs just as fast and comprehensively as the RNAi technique has.RNAi technique has.

Nature, News. Allison AbbottNature, News. Allison Abbott

RNAi studies in PubMedRNAi studies in PubMed

050

100150200250300350400450500

1 2 3 5 10

RNAi

siRNA

Years before 2007

Nu

mb

er o

f P

ub

lica

tio

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Ways to induce silent phenotypesWays to induce silent phenotypes

Timmons and Fire showed that feeding dsRNA works!Timmons and Fire showed that feeding dsRNA works!Reversible and gene-specific effects…Reversible and gene-specific effects…

Ways to induce silent phenotypesWays to induce silent phenotypes

Tabarra, Grishok, and Mello in 1998 demonstrated that Tabarra, Grishok, and Mello in 1998 demonstrated that soaking in dsRNA also works!soaking in dsRNA also works!

Nomarski image showing embryos produced by a wild-type mother treated with pos-1 RNAi by soaking. All except one embryo (arrow) show the distinctive pos-1 embryonic arrest with no gut, no body morphogenesis, and extra hypodermal cells

pos-1 encodes a CCCH-type zinc-finger protein; maternally provided POS-1 is essential for proper fate specification;

Mechanisms revealedMechanisms revealed

25bp species of dsRNA found in plants with co-25bp species of dsRNA found in plants with co-suppression suppression [Hamilton and Baulcombe, 1999][Hamilton and Baulcombe, 1999]

Sequence similar to gene being suppressedSequence similar to gene being suppressed

Drosophila: long dsRNA “triggers” processed Drosophila: long dsRNA “triggers” processed into 21-25bp fragments into 21-25bp fragments [Elbashir et al., 2001][Elbashir et al., 2001]– Fragments = short interfering RNA (siRNA)Fragments = short interfering RNA (siRNA)– siRNA necessary for degradation of targetsiRNA necessary for degradation of target

RNAi: two phasesRNAi: two phases

InitiationInitiation– Generation of mature siRNA or miRNAGeneration of mature siRNA or miRNA

ExecutionExecution– Silencing of target geneSilencing of target gene– Degradation or inhibition of translationDegradation or inhibition of translation

RNAi illustrated RNAi illustrated

How does RNAi work?How does RNAi work?

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siRNA biogenesissiRNA biogenesisDicer Dicer (type III RNAse III) (type III RNAse III) cleaves long dsRNA cleaves long dsRNA into siRNA 21-25ntinto siRNA 21-25nt dsRNA from exogenous dsRNA from exogenous sourcessources– Symmetric 2nt 3’ overhangs, 5’ phosphate groupsSymmetric 2nt 3’ overhangs, 5’ phosphate groups– Evidence for amplification in C. elegans and plantsEvidence for amplification in C. elegans and plants

RNA Induced Silencing Complex RNA Induced Silencing Complex (RISC)(RISC)

RNAi effector complexRNAi effector complex

Preferentially incorporates one strand of Preferentially incorporates one strand of unwound RNA [Khvorova et al., 2003]unwound RNA [Khvorova et al., 2003]– AntisenseAntisense

How does it know which is which?How does it know which is which?– The strand with less 5’ stability usually incorporated The strand with less 5’ stability usually incorporated

into RISC [Schwarz et al., 2003]into RISC [Schwarz et al., 2003]

siRNA designsiRNA design

Mittal, 2004

Custom-made siRNAsCustom-made siRNAs

siRNA librariessiRNA libraries

Generation of a feeding cloneGeneration of a feeding clone

Tuschl, 2003

siRNA librariessiRNA libraries

Result: 16 757 bacterial strainsResult: 16 757 bacterial strains

86.3% of predicted genes with RNAi 86.3% of predicted genes with RNAi phenotypes assignedphenotypes assigned

Assayed Phenotypes: ExamplesAssayed Phenotypes: Examples

Emb – embryonic lethalEmb – embryonic lethalSte – sterileSte – sterileGro – slow growthGro – slow growthAdl – adult lethalAdl – adult lethalLvl – larval lethalityLvl – larval lethalityLva – larval arrestLva – larval arrestBmd – body Bmd – body morphological defectsmorphological defects

Unc – uncoordinatedUnc – uncoordinated

Clr – clearClr – clear

Prz – paralyzedPrz – paralyzed

Lon – longLon – long

Mlt – moulting defectsMlt – moulting defects

Egl – egg laying defectsEgl – egg laying defects

Him – high incidence of Him – high incidence of malesmales

Endogenous RNAi-miRNAEndogenous RNAi-miRNA

We have hundreds of We have hundreds of different genes that different genes that encode small RNA encode small RNA (collectively, microRNA) (collectively, microRNA) whose precursors can whose precursors can form double-stranded form double-stranded RNA. These can RNA. These can activate the RNA activate the RNA interference process and interference process and thus switch off the thus switch off the activity of various genes activity of various genes with matching segments.with matching segments.First miRNA is lin-4First miRNA is lin-4

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More miRNAsMore miRNAs

No other miRNAs No other miRNAs found for 7 years!found for 7 years!– Second miRNA – let-7 Second miRNA – let-7

[Reinhart et al., 2000][Reinhart et al., 2000]– Non coding, 21nt RNANon coding, 21nt RNA– Regulates lin-14 in Regulates lin-14 in

same way as lin-4same way as lin-40

10

20

30

40

50

60

70

1 2 3 5 10

miRNA

Years before 2007N

um

ber

of

Pu

bli

cati

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Defense Against VirusesDefense Against Viruses

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Indeed, Baulcombe, Vance, and others have shown that, in the Indeed, Baulcombe, Vance, and others have shown that, in the continuing evolutionary war to survive and reproduce, plant viruses have continuing evolutionary war to survive and reproduce, plant viruses have evolved genes that enable them to suppress silencing.evolved genes that enable them to suppress silencing.

Mammalian RNAiMammalian RNAi

McManus and Sharp, 2002

Getting Around the ProblemGetting Around the Problem

siRNA (21-22nt) mediate mammalian siRNA (21-22nt) mediate mammalian RNAiRNAi– Introducing siRNA instead of dsRNA prevents Introducing siRNA instead of dsRNA prevents

non-specific effectsnon-specific effects

Some applications of RNAiSome applications of RNAi

TherapyTherapy– Candidate genes, drug discovery, and therapyCandidate genes, drug discovery, and therapy

Genome-wide RNAi screensGenome-wide RNAi screens– Gene functionGene function– Candidate genes and drug discoveryCandidate genes and drug discovery

Systems biologySystems biology– Models of molecular machinesModels of molecular machines

Drugging the wormsDrugging the worms

AGING, CANCER, NEURODEGENERATION, AGING, CANCER, NEURODEGENERATION, NEUROBIOLOGYNEUROBIOLOGY

Genome-wide ScreensGenome-wide Screens

Early Embryonic PhenotypesEarly Embryonic Phenotypes

Osmotic integrityOsmotic integrity

Passage through Passage through meiosismeiosis

Entry into interphaseEntry into interphase

Cortical dynamicsCortical dynamics

Nuclear appearanceNuclear appearance

Sister chromatid Sister chromatid separationseparationAsymmetry of divisionAsymmetry of divisionCytokinesisCytokinesisPace of developmentPace of developmentCentrosome attachmentCentrosome attachment

Genome-wide RNAiGenome-wide RNAiA total of 19,075 genes were targeted by dsRNAs.More than 40,000 time-lapse microscopy recordings, scored and annotated.

Genome-wide RNAiGenome-wide RNAi

Only 11% genes showed detectable RNAi Only 11% genes showed detectable RNAi phenotypephenotypeBetween 600-800 genes are required for early Between 600-800 genes are required for early embryogenesis.embryogenesis.

Systems Biology and RNAiSystems Biology and RNAi

Cellular systems act as networks of interacting Cellular systems act as networks of interacting components (genes, RNA, protein, metabolites,components (genes, RNA, protein, metabolites,…).…).

Genome-wide RNAi screens offers the potential Genome-wide RNAi screens offers the potential for revealing functions of each protein.for revealing functions of each protein.

Combining RNAi screen data with other Combining RNAi screen data with other highthroughput data (e.g., protein-protein highthroughput data (e.g., protein-protein interaction, mRNA expression profiling) leads to interaction, mRNA expression profiling) leads to understanding of the organization of the cell understanding of the organization of the cell system.system.

‘‘Tis true, There’s magic in the web of it.Tis true, There’s magic in the web of it.

William Shakespeare (1602-4)William Shakespeare (1602-4)

The Future: Integrative BiologyThe Future: Integrative Biology

Networks of Early EmbryogenesisNetworks of Early Embryogenesis

Protein-protein interaction dataset:Protein-protein interaction dataset: binary physical binary physical interactions between 3,848 interactions between 3,848 C. elegansC. elegans proteins proteins

Transcriptome dataset:Transcriptome dataset: expression profiling similarity expression profiling similarity above a given threshold among genes in the networkabove a given threshold among genes in the network

Phenotypic dataset:Phenotypic dataset: phenotypic similarity above phenotypic similarity above another threshold of 661 early embryogenesis genes. another threshold of 661 early embryogenesis genes. RNA interference (RNAi) phenotypic signature consisting RNA interference (RNAi) phenotypic signature consisting of a vector describing specific cellular defects in early of a vector describing specific cellular defects in early embryogenesis.embryogenesis.

Systems Biology Approach:Systems Biology Approach:Three networks in oneThree networks in one

Does it make sense to combine?Does it make sense to combine?

The embryogenesis networkThe embryogenesis network

The aesthetics of natural science and The aesthetics of natural science and mathematics is at one with the aesthetics mathematics is at one with the aesthetics of music and paintings both inhere in the of music and paintings both inhere in the discovery of a partially concealed pattern.discovery of a partially concealed pattern.

Herbert Simon (1996)Herbert Simon (1996)

Herbert Alexander Simon (June 15, 1916 – February 9, 2001), an American political scientist and polymath; one of founding fathers of Artificial Intelligence, information processing, decision-making, and complex systems, the first to analyze the architecture of complexity and to propose a preferential attachment mechanism to explain power law distributions.

1995-Guo & Kemphues1995-Guo & Kemphues

1995-Guo & Kemphues1995-Guo & Kemphues

A book: Critical Mass by Phillip BallA book: Critical Mass by Phillip Ball

How one thing leads to otherHow one thing leads to otherHe details the development He details the development of key concepts in of key concepts in contemporary physics, such contemporary physics, such as self-organization, and as self-organization, and chaos; Next, he shows how chaos; Next, he shows how social scientists apply these social scientists apply these concepts to the study of concepts to the study of human organization. human organization. the relationship between the relationship between global phenomena and local global phenomena and local actions. actions.

The reader will appreciate the orderliness The reader will appreciate the orderliness of the lines… and he will see how this of the lines… and he will see how this orderliness points to the existence of a orderliness points to the existence of a fundamental governing principle.fundamental governing principle.

George Kingley Ziph (1949)George Kingley Ziph (1949)

Links to endogenous dsRNALinks to endogenous dsRNA

1999-Hamilton & Baulcombe1999-Hamilton & Baulcombe

Hypothesis: Hypothesis: Antisense RNA Antisense RNA complementary to complementary to the target mRNA the target mRNA may be too short to may be too short to observe so is easy to observe so is easy to miss.miss.Tomato lines Tomato lines transformed with transformed with ACO cDNA under ACO cDNA under the 35S promoter.the 35S promoter.

1999-Hamilton & Baulcombe1999-Hamilton & Baulcombe

1999-Hamilton & Baulcombe 1999-Hamilton & Baulcombe concluded:concluded:

Discovery ProjectDiscovery Project

C. Elegans as a Disease ModelC. Elegans as a Disease ModelAGING, NEURODEGENERATION, NEUROBIOLOGYAGING, NEURODEGENERATION, NEUROBIOLOGY– Longevity model: daf-2 mutants and RNAi showed that reduced insuling signaling Longevity model: daf-2 mutants and RNAi showed that reduced insuling signaling

increases resistance oxincreases resistance ox

Discovery of miRNADiscovery of miRNA

Discovery of the first miRNA, lin-4Discovery of the first miRNA, lin-4– Non-coding, 22nt RNANon-coding, 22nt RNA– Identified in screen for defects in timing of larval developmentIdentified in screen for defects in timing of larval development

lin-4 mutation – ectopic larval stage 1-like cell divisions lin-4 mutation – ectopic larval stage 1-like cell divisions at later stagesat later stageslin-14 mutations – reciprocal phenotype, same regulatory lin-14 mutations – reciprocal phenotype, same regulatory pathway as lin-4pathway as lin-4– lin-4 negatively regulates lin-14 translationlin-4 negatively regulates lin-14 translation– lin-4 partially complementary to conserved sites in lin-14 3’UTR lin-4 partially complementary to conserved sites in lin-14 3’UTR

[Lee et al., 1993][Lee et al., 1993]– Required for negative regulation of lin-14Required for negative regulation of lin-14– lin-4 binds these siteslin-4 binds these sites

More miRNAsMore miRNAs

miRNA is in other organisms?miRNA is in other organisms?– Let-7 Homologs were easily detected Let-7 Homologs were easily detected

[Pasquinelli et al., 2000][Pasquinelli et al., 2000]Drosophila, sea urchins, mice, humans...Drosophila, sea urchins, mice, humans...

Indicates RNAi general conserved mechanismIndicates RNAi general conserved mechanism

RNAi in other genesRNAi in other genes

Hlh-1: myogenic regulatory factor (MRF) Hlh-1: myogenic regulatory factor (MRF) subgroup of bHLH proteins; HLH-1 activity is subgroup of bHLH proteins; HLH-1 activity is required during embryonic development for the required during embryonic development for the proper differentiation and function of body wall proper differentiation and function of body wall muscle cellsmuscle cells

RNAi in other genesRNAi in other genes

Unc-54: encodes a muscle myosin class II heavy chain Unc-54: encodes a muscle myosin class II heavy chain (MHC B) required for locomotion and egg-laying(MHC B) required for locomotion and egg-laying

Defense against transposonsDefense against transposons

RNAi may also help keep the transposable RNAi may also help keep the transposable elements that litter genomes from jumping elements that litter genomes from jumping around and causing harmful mutations. around and causing harmful mutations. Plasterk's team and Mello, Fire, and their Plasterk's team and Mello, Fire, and their colleagues found that mutations that colleagues found that mutations that knocked out RNAi in C. elegans led to knocked out RNAi in C. elegans led to abnormal transposon movements. abnormal transposon movements.

I have endeavored to show that it is the I have endeavored to show that it is the peculiar function of physical science to peculiar function of physical science to lead us to the confines of the lead us to the confines of the comprehensible, and to bid us behold it comprehensible, and to bid us behold it and receive it in faith, till such time as the and receive it in faith, till such time as the mystery shall open.mystery shall open.

James Clerk Maxwell (1856)James Clerk Maxwell (1856)

Rethinking of the antisense dogmaRethinking of the antisense dogma

When 19th century Scottish physicist James Clerk Maxwell was faced with predicting the behavior of gases made up of trillions of rapidly moving molecules, he borrowed from social scientists the

mathematical tools of statistics and successfully applied them to the problem.