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Mammalian RNAi pathways Michael T. McManus MIT 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 and then introduced into cells . dsRNA. nucleus.

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slide1
Mammalian RNAi pathways

Michael T. McManus

MIT Center for Cancer Research

slide2
what is RNA interference?

•RNAi is a way to silence gene expression

•to perform RNAi, dsRNA homologous

to the targeted gene is made and

then introduced into cells

dsRNA

nucleus

•any mRNA with high sequence homology

to the dsRNA may be silenced

rnai a tool for inhibiting gene expression in vivo
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

practical aspects of RNAi
slide5
How does RNAi work?

RNAi works postranscriptionally……..

in key two steps!

slide6
step one:

34

27

21

20

16

short-interfering RNA

processing the dsRNA into 21-23 nt fragments

Tuschl, 2001

slide8
19 nt duplex

2 nt 3’ overhangs

siRNAs have a defined structure

slide9
step two:

the antisense strand of the siRNA guides cleavage

Tuschl, 2002

slide10
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
slide12
P

P

interferon

production

PKR

eiF2a

P

apoptosis

Blockage of protein synthesis

Mammals exhibit potent responses to dsRNA

dsRNA

cell death

slide13
smaller RNAs can escape the PKR pathway

recall that siRNAs are

intermediate effectors

In the RNAi pathway

siRNAs are not recognized

by the PKR!

slide14
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?

slide15
how to get siRNAs into the T-cells

cationic lipids, calcium phosphate, etc.

dead cells

T-cell

receptor-dependent transport,

endocytosis, etc.

no silencing

electroporation

slide16
develop an assay quantitative on the single-cell level

T-cell

CD8

flow

cytometry

detector

CD4

fluorescent antibodies detect

expression on the single cell level

slide18
CD8 mRNA

5’ UTR

CD8 ORF

3’ UTR

CD8 siRNAs

CD4 mRNA

5’ UTR

CD4 ORF

3’ UTR

CD4 siRNAs

can any region of the mRNA be targeted with siRNAs?

+

+

McManus, 2002

slide19
5’

3’

PB2

PB1

PA

NP

M

NS

siRNA

UTR

No inhibition

Coding sequence

Partial inhibition

Strong inhibition

Influenza mRNA target-sites

Ge, 2003

slide20
% cells silencing CD8

cell mass

how long does the RNAi response last?

McManus, 2002

slide21
miniconclusion

RNAi works by target degradation of the mRNA

RNAi creates knock-downs, not knockouts!

not every siRNA works

RNAi via siRNAs is transient,

lasting ~3-6 cell doublings

slide22
establishing long-term RNAi

Let the cell make the siRNA for you!

slide23
CD8 hairpin RNAs

McManus, 2002

slide24
hairpin siRNAs

McManus, 2002

slide25
stable mammalian RNAi

Within a three month window:

Brummelkamp et al: Science

Yu et al: PNAS

Miyagishi et al: Nature Biotech

Sui et al: PNAS

Sook Lee et al: Nature Biotech

Zeng et al: Mol Cell

Paddison et al: Genes Dev

Paul et al: Nature Biotech

McManus et al:RNA

slide27
lentiviral construct for siRNAs

Rubinson et al Nature Genetics, 2003

slide28
Lentiviral CD8 knockdown

Rubinson et al Nature Genetics, 2003

slide29
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
functional silencing of genes in ES cell-derived mice by lentivirus-induced RNAi

Rubinson et al Nature Genetics, 2003

slide31
mini-conclusion

Although silencing by siRNAs is transient, vectors

can be made to express siRNAs in cells

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

RNAi silencing can be transmitted through the germline

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