RNA Silencing, mechanism and applications

The Central Dogma of Molecular Biology

There is “life” outside protein-coding genes

• There are approximately 70.000 gene predictions for the Human genome

…But the estimated number of protein coding genes is 21,561

• It is estimated that 98% of the transcriptional output of the human genome represents RNA that does not encode protein

RNA Silencing, mechanism and applications

2. The role of small RNAs in gene regulation

3. Mechanisms of RNA interference

4. Biological role of RNA silencing

5. Applications of RNA silencing

6. miRNAs

1. History and definitions

Posttranscriptional gene silencing (PTGS)Posttranscriptional gene silencing (PTGS)

Classical key experiment in petunia:

• Artificially introduced 2 nd CHS gene

• Expected: more intense flowers

Real outcome: Variable phenotypes, including white flowers

• Key enzyme for flower pigmentation:

chalcone synthase (CHS)

van der Krol et al., Plant Cell 2:291 (1990) + Plant Mol Biol 14:457 (1990).

Effects of expression of CHS sense and antisense RNA on flower pigmentation in Petunia

•The silencing of an endogenous

gene due to the presence of a

homologous transgene or virus.

•Co-suppression can occur at the

transcriptional or posttranscriptional

level.

CO-SUPPRESSION

Key experiment on RNA interference (RNAi)

Nature 391, 806 - 811 (1998);Nature 391, 806 - 811 (1998);

Injection of ds RNA to C. elegans causes

gene-specific silencing

Injection of ds RNA to C. elegans causes

gene-specific silencing

New term:

RNA interference (RNAi)

New term:

RNA interference RNA interference (RNAi)(RNAi)

Entrez PubMed Search Terms: RNAi or siRNA or miRNA

Number of Publications

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23.000

RNA silencing

• RNA mediated impairment of transcription- TGS-methylation (shRNAs, rasiRNAs and siRNAs?)

• RNA mediated impairment of translation-PTGS (siRNAs and some miRNAs) and -translational arrest (miRNAs)

Small RNA classification

small RNAs

Sequence specific RNAdegradation

Post-Transcriptinalgene silencing

Short interfering

RNA (siRNA)

Inhibition of translation

Gene regulation

microRNA miRNA

RNA mediated methylation

rasiRNAs, endo-siRNAs etc.

Regulation of the Epigenome

Various types of suppression

Suppression of transposable elements

stress response etc

piRNAtasiRNA

natsiRNA

2. The role of small RNAs in gene regulation

1. History

RNA σίγηση, ένας μηχανισμόςρύθμισης και άμυνας

short interfering RNA (siRNA)short interfering RNA (siRNA)

NN 5’

5’

ca 21 NucleotidesNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNN

3’

3’

short interfering RNA (siRNA)short interfering RNA (siRNA)

NNca 21 Nucleotides

NNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNN

3’

3’

- N N N

- N

N -

N N N -two 3’ protruding

unpaired nucleotides

5’ P -- P 5’

5’ terminal phosphates

PTGS / RNAi inductionPTGS / RNAi induction

Target RNA

antisense RNA

RNA quant( threshold )

“aberrant”RNA

Ectopic

pairing

ds

RNARdRP?

RdRP? ?

Basic Model for the MechanismBasic Model for the Mechanism

Target RNA

antisense RNA

RNaseRNase IIIIII--type enzyme type enzyme DicerDicer

Target RNA

antisense RNA

RNaseRNase IIIIII--type enzyme type enzyme DicerDicer

Basic Model Basic Model

short short interfering interfering

RNARNA( siRNA )( siRNA )

Basic Model Basic Model

short interfering RNAshort interfering RNA

siRNAs play an essential role in RNA interference

They are mediators as well as indicators of posttranscriptional gene silencing (PTGS)

Integration of siRNA to RISCIntegration of siRNA to RISC

N

N

N

N

N

N

N

N

N

N

N

N - P 5’

P -5’

ca 21 Nucleotides

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Incorporation in

RNA-induced silencing complex

(RISC complex)

Degradation of Target RNADegradation of Target RNA

targetRNAs

cleavedtargetRNAs

Degradation of target RNA: major playersDegradation of target RNA: major players

DicerDicer RISCRISC

siRNAsiRNA

Dicer

• Dicer generates RNAs with 2 nt 3’overhang and 5’ phosphorylated terminus, both required for activity

• Fly Dicer requires ATP, human may not

Bact.RNase III

Class II: DroshaClass III dicer

ENZYME STRUCTURE – RNase III

Different organisms have different numbers of Dicers

Mello CC, Conte D; Nature 431:338, 2004

Transgenic GFP expressing N.benthamiana is our system

Silencing in plants is a non-cell autonomous event

The Mechanism of RNA silencing is to a certain extent conserved

between eucaryots

Testing plant extracts for their silencing potentialTesting plant extracts for their silencing potential

mRNA

si RNAGFP non-silenced GFP silenced

RNA extract

GFP-expressing

C.elegansRNA extract

RNA silencing pathways in fungi

RNA silencing pathways in fungi

AS, ascomycete; BS, basidiomycetes; ZY, zygomycete; OO, oomycete; AC, Aspergillus clavatus; AFl, Aspergillusflavus; AFu, Aspergillus fumigatus; AN, Aspergillus nidulans; AO, Aspergillus oryzae; AN, Aspergillus terreus; CA, Candida albicans; CC, Coprinus cinereus; CL, Candida lusitaniae; CN, Cryptococcus neoformans; CT, Candidatropicalis; FG, Fusarium graminearum; FO, Fusarium oxysporum; FV, Fusarium verticillioides; HC, Histoplasmacapsulatum; MO, Magnaporthe oryzae; NC, Neurospora crassa; PC, Phanerochaete chrysosporium; PG, Pucciniagraminis; PI, Phytophthora infestans; RO, Rhizopus oryzae; SC, Saccharomyces cerevisiae; SN, Stagonosporanodorum; SP, Schizosaccharomyces pombe; UM, Ustilago maydis; AT, Arabidopsis thaliana.

In fungi there seems to be an avoidance of RNAi when “killer”

virus is present

2. The role of small RNAs in gene regulation

3. Mechanisms of RNA interference

4. Biological role of RNA silencing

1. History and definitions

RNA σίγηση, ένας μηχανισμόςρύθμισης και άμυνας

Biological role of silencing

Protection against invasive RNAsespecially RNA viruses

Silencing of transposable elements

Regulation of expression of endogenous genes

2. The role of small RNAs in gene regulation

3. Mechanisms of RNA interference

4. Biological role of RNA silencing

5. Applications of RNA silencing

1. History

RNA σίγηση, ένας μηχανισμόςρύθμισης και άμυνας

Applications of RNA silencing

• Functional analysis of genes

• Sequence specific suppression of unwanted genes

• Generation of plants resistant to viruses

Functional analysis of gene by reverse genetics

1. Identification of gene sequence

2. Reverse genetics:-find copy number/possibly locus (Arabidopsis)-charachterise time and space of expression-find gene function a. overexpressionb. suppression (knock-down, knock-out)

RNAi induced at the level of siRNAs

Direct application of siRNA

(a) chemically synthesized(b) enzymatically synthesized

delivery via short hairpin RNAs (shRNA)

Vector-mediated RNAi (DNA or virus)

Strategies of expressing double-stranded RNA

Pol II and Pol III (type III) e.g U6 prom

Promoter spacer/intron

terminator

AAA

alternative: tandem promoters, opposing promoters

Hairpin induced gene silencing of CP gene

RB

35S

(+) CPfrag.

spacer nptII

LB(-)CP frag.

ter

~ ~~ ~ ~Dicer

~ ~~ ~ ~~ ~~ ~ ~

RISC

~ ~~ ~ ~

RNA σίγηση, ένας μηχανισμόςρύθμισης και άμυνας

2. The role of small RNAs in gene regulation

3. Mechanisms of RNA interference

4. Biological role of RNA silencing

5. Applications of RNA silencing

6. miRNAs

1. History and definitions

MicroRNAs

• short (20-25nt) RNA molecules• translated as precursor RNA

molecule• post transcriptional gene regulation• target mRNA for cleavage or

translational repression• very abundant class of genes

The processing pathway of siRNA & miRNA

from Dykxhoorn et al., (2003) Nature Reviews 4, 457-466.

Mechanism of miRNA suppression of gene expression

• Transcription• mRNA degradation• Translational repression

– 1 Initiation– 2 Elongation– 3 Termination– 4 Release

• Co-translational degradation of the nascent peptide

Cullen Nature Immunology 7:563 2006

Published by AAASJ. Liu et al., Science 305, 1437 -1441 (2004)

Identification of Argonaute 2 as Slicer in humans

miRNA• The miRNA are endogenous small RNA guides that

repress the expression of target genes.• Differ from siRNA in biogenesis not in functions,

although mechanisms can be different. mRNA cleavage when complementarity is extensive, repress translation when not.

• lin-4 mutant worms had defects in timing of cell division. Encodes a small RNA that binds to and silenced lin-14 message.

• Lin-14 mRNA levels do not decline, but that may not always be the case.

• let-7 also found in other species.

miRNA

• Many miRNA are embedded in introns of protein encoding genes and are transcribed together with host genes.

• miRNA can be expressed in developmentally tissue specific fashion but may not be expressed in tissues where putative target sequences are.

Du, T. et al. Development 2005;132:4645-4652

The structure of human pri-miRNAs

Useful plant miRNA properties

• (at least some) miRNAs are conserved between species

• most precursor sequences are not conserved

• precursor structure is somewhat conserved

• mature miRNA always cut from 1 arm of RNA helix in precursor

• plant precursors are more complex

Plasterk Cell 124:877, 2006

PLoS, 39 (3): 404-418

Summary of plant miRNA/target recognition

1. Perfect match for positions 2-12

2. No more than 2 consecutive mismatches at 3’

3. Free energy at least 72% that of perfect match

Esquela-Kerscher et al. Nature Reviews Cancer 6, 259–269 (April 2006) | doi:10.1038/nrc1840

MicroRNAs can function as tumour suppressors and oncogenes

And yet more roles for small RNAs…

RNA σίγηση, ένας μηχανισμόςρύθμισης και άμυνας

2. The role of small RNAs in gene regulation

3. Mechanisms of RNA interference

4. Biological role of RNA silencing

5. Applications of RNA silencing

6. miRNAs

1. History and definitions

7. RNA-mediated epigenetic changes

Herve Vaucheret Genes Dev. 2006; 20: 759-771

Detection of methylation in DNADetection of methylation in DNA

HpaII CCGG

MspII CCGG

CMG

CMNG

HaeIII GGCC CMNN

CCMGG

CMCGG

GGCMC

Restrictionenzyme

cut no cut detection of

or bisulphite sequencing in combination with PCR

Discovery of DNA-dependent Pol IV in plantsDiscovery of DNA-dependent Pol IV in plants

Nature Reviews Genetics 4; 29-38 (2003); doi:10.1038/nrg982EXPLORING PLANT GENOMES BY RNA-INDUCED GENE SILENCING