Dogmatic View of Gene Expression

DNA ProteinRNA

Post-transcriptional Control:Quantitative Control:

Levels of mRNA not proportional to levels of mRNA synthesized or protein produced

Qualitative Control: More than one protein from a single gene

(e.g. Differential RNA Processing or RNA editing)

An “RNA-Centric” View ofGene Expression

RNADNA Protein

RNA: A Diverse Class of Molecules

RNADNA

Non Coding RNAs: ‘RiboRegulators’

(~97% of RNAs Present in Human Cells are Non-Coding)

rRNAtRNA

snRNAssnoRNAsGuide RNA

Catalytic:RibozymesTelomerase

Introns5’ UTR3’ UTR

VaultY RNAs7SK

Xist, H19

MicroRNAs

Viral RNAsRetrotransposons

Non Coding RNAs:

SnoRNAs • Large Family

• Intron-encoded

• Guide RNA Modification

Non Coding RNAs:

Telomerase RNA • Component of telomerase

• Provides template for telomere synthesis

• Role in Cancer and Aging

RNA-Mediated Gene Silencing

Post-transcriptional Gene Silencing (PTGS) or RNA Interference (RNAi)

Transcriptional Gene Silencing (TGS)(RNA-dependent DNA Methylation)

Gene Silencing By MicroRNAs

Discovery of RNAi

Double-stranded RNA

inject

C. elegans

Neg. control Uninjected

Antisense RNA dsRNA

sense

antisense

Nature 1998 391:806-811

Mex-3 mRNA detection in embryos by in situ hybridization

Remarkable Properties of RNAi• dsRNA (not ssRNA) is interfering agent

• Sequence-specific loss of mRNA and protein

• Effective against exons not introns

• Potent (few dsRNA molecules/cell effective)

• Persistent (affects next generation)

• Effects can cross cell barriers (feed, soak)

Mechanism of RNAi: Gene Silencing directed by ~22nt RNAs

dsRNA

~22nt siRNAs

targetmRNA

secondary siRNAs

amplification

processing

degradation

recognition

copying+

processing

spreading

Gene Silencing Factors

C. elegansDrosophila Arabidopsis

amplification

processing

degradation

recognition

copying+

processing

spreadingDICERDCR-1CAF

AGO2RDE-1AGO1R

ISC

SID-1RRF-1SDE-1/SGS-2

RDE-4

VIGCG1800

Fmr1

RNAi in Mammalian Cells• Long dsRNA triggers global (non-specific) gene-silencing (i.e. interferon response)• Breakthrough: Short dsRNA (~22 nt) induces RNAi

Nature 2001 411: 494-498

Silencing of lamin proteins in human cells by dsRNA transfection

RNA-Mediated Gene Silencing

RNA Interference‘Cosuppression’ by transgenes in plants

‘Quelling’ in FungiTranscriptional Gene Silencing (TGS)

Common Trigger:

RNAi Applications

GENETIC TOOL

GENE THERAPY

Probing Gene Function

Combat Viral InfectionTreat Genetic Diseases

(New expression strategies)

What is the Purpose of RNA-Mediated

Gene Silencing?

Proposed Biologic Roles

• Antiviral Defense• Suppress Transposon Activity• Response to Aberrant RNAs

• Gene Regulation (e.g. MicroRNAs)

‘Immune System’ of the Genome

RNA-Mediated Gene Silencing

Science 2002 296:1263-1265

MicroRNAs: Expanding Family of ‘RiboRegulators’

• lin-4 and let-7 RNAs (from worm) were first examples• Also known as stRNAs (small temporal RNAs)• Regulate expression of proteins and developmental timing• Tip of the iceberg………..MicroRNAs are everywhere!

RNAi by siRNAs Developmental regulationby stRNAs (µ RNAs)

processing~22nt

siRNAs~22ntlin-4

processing

target recognition

target recognition

mRNA

lin-14mRNA

lin-41mRNA

3’UTR

3’UTR

degradation

~22ntlet-7

Translational repression