Mammalian RNAi pathways

Michael T. McManusMIT Center for Cancer Research

what is RNA interference?

• RNAi is a way to silence gene expression

• to perform RNAi, dsRNA homologous to the targeted gene is made andthen introduced into cells

• any mRNA with high sequence homology to the dsRNA may be silenced

nucleus

dsRNA

RNAi: a tool for inhibiting gene expression in vivo

• C. elegans (Fire et al., 1998)• Drosophila (Carthew et al., 1998) • Planaria (Newmark et al., 1998)• Trypanosomes (Ullu et al., 1998) • Hydra (Lohmann et al., 1999)• Zebrafish (Wargelius et al., 1999)• Mice (Wianny & Zernicka-Goetz, 2000)• “cosuppression” in plants• “quelling” in Neurospora

practical aspects of RNAi

• biological research– defining gene function (gene knockout)

• C. elegans genome RNAi projects

– defining biochemical pathways• microarray screening of RNAi knockouts

• therapeutic treatment– cancer

– viral infection

– parasitic infection

How does RNAi work?

RNAi works postranscriptionally……..

in key two steps!

processing the dsRNA into 21-23 nt fragments

3427212016

short-interfering RNA

QuickTime™ and aGIF decompressor

are needed to see this p icture.

step one:

Tuschl, 2001

Dicer contains two RNAse III domains

siRNAs

long dsRNA

siRNAs have a defined structure

19 nt duplex

2 nt 3’ overhangs

Tuschl, 2002

the antisense strand of the siRNA guides cleavagestep two:

RNAi silencing complex

• may be associated with translating ribosomes

• active RNAse enzyme not yet identified

• may participate in endogenous pathways that silence genes via translational repression

siRNA

Model for RNAi

Mammals exhibit potent responses to dsRNA

PKR

PP

P

eiF2

dsRNA

Blockage of protein synthesis

interferonproduction

cell deathapoptosis

smaller RNAs can escape the PKR pathway

siRNAs are not recognized by the PKR!

recall that siRNAs are intermediate effectorsIn the RNAi pathway

need to further characterize mammalian RNAi

how long does it last?

how much dsRNA is required?

can any region of a gene be effectively targeted?

T-cell

how to get siRNAs into the T-cells

electroporation

receptor-dependent transport,endocytosis, etc.

cationic lipids, calcium phosphate, etc.

dead cells

no silencing

T-cellCD4

CD8flow

cytometrydetector

fluorescent antibodies detect expression on the single cell level

develop an assay quantitative on the single-cell level

transfection of plasmids and siRNAs

McManus, 2002

5’ UTR CD8 ORF 3’ UTR

CD8 siRNAs

CD8 mRNA

5’ UTR CD4 ORF 3’ UTR

CD4 mRNA

CD4 siRNAs

can any region of the mRNA be targeted with siRNAs?

+

+

McManus, 2002

PB2

PB1

PA

NP

M

NS

UTR

5’ 3’

Coding sequence

No inhibition

Partial inhibition

Strong inhibition

siRNA

Influenza mRNA target-sites

Ge, 2003

how long does the RNAi response last?

0.00

20.00

40.00

60.00

80.00

100.00

120.00

0 10 1000 100000

cell mass

% c

ells

sile

nci

ng

CD

8

McManus, 2002

miniconclusion

RNAi via siRNAs is transient, lasting ~3-6 cell doublings

RNAi creates knock-downs, not knockouts!

not every siRNA works

RNAi works by target degradation of the mRNA

establishing long-term RNAi

Let the cell make the siRNA for you!

CD8 hairpin RNAs

McManus, 2002

hairpin siRNAs

McManus, 2002

stable mammalian RNAi

Within a three month window:

McManus et al:RNA

Brummelkamp et al: Science

Paul et al: Nature Biotech

Sui et al: PNAS

Yu et al: PNAS

Sook Lee et al: Nature Biotech

Miyagishi et al: Nature Biotech

Paddison et al: Genes Dev

Zeng et al: Mol Cell

lentiviral construct for siRNAs

Rubinson et al Nature Genetics,

2003

Lentiviral CD8 knockdown

Rubinson et al Nature Genetics,

2003

stable 14-fold CD8 knockdown by lentivirus siRNAs

Rubinson et al Nature Genetics,

2003

functional silencing of genes in ES cell-derived mice by lentivirus-induced RNAi

Rubinson et al Nature Genetics,

2003

mini-conclusion

RNAi knock-down mice can be generated in <30 days

RNAi silencing can be transmitted through the germline

Although silencing by siRNAs is transient, vectors can be made to express siRNAs in cells