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The mechanism of RNAi. by Renchang. Guo S et al, a gene required for establishing polarity in C. elegans embryos, encoding a putative Ser/Thr kinase that is asymmetrically distributed, cell, 1995, 81:611. Fire Andrew, et al, Potent and specific genetic interference

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Guo S et al,

a gene required for establishing polarity in C. elegans embryos, encoding a putative Ser/Thr kinase that is asymmetrically distributed,

cell, 1995, 81:611


Fire Andrew, et al,

Potent and specific genetic interference

by double-strand RNA in Caenorhabditis

elegans.

Nature, 1998, 391:806-811


RNAi is characteristic of:

Extreme efficiency: a few trigger dsRNA

molecules suffice to inactivate a continuously

transcribed target mRNA for long periods

of time;

Long-lasting: the inactivation persists through

cell division, spreads to untreated cells and

tissues of plants, and is even inhetited by

subsequent generations of nematodes.


Bernstein, 2001

Hamilton, 1999

?

Cogoni, 1999

Smardon, 2000

Dalmay, 2000

Lipardi, 2001

Nykanen, 2001


Sijen, 2001


Hamilton, A.J., et al,

A species of small antisense RNA in

posttranscriptional gene silencing in plants.

1999, Science, 286, 950-952.


Bernstein E, et al,

Role for a bidentate ribonuclease in the

initiation step of RNA interference.

Nature, 2001, 409:363-366

Dicer: a dsRNA-specific endonuclease

responsible for processing of long

targeting dsRNA into siRNAs.


Cogoni, et al, Gene silencing in Neurospora crassa reqiures a protein homologous to RNA-dependent RNA polymerase, Nature 399, 166-169

QDE-1 N. crassa


Ego 1 c elegans

Smardon. A., et al, EGO-1 is related to RNA-directed RNA polymerase and functions in gern-line development and RNA interference in C. elegans. 2000, curr.Biol. 10, 169-178.

EGO-1, C. elegans



Material: Arabidopsis, transgenes

PVS:GFP: responsible for initiation of PTGS

GFP: reporter of silencing

Four genetic loci are requried for PTGS

Sde Silencing defective

Sde1, Sde2, Sde3, Sde4


This paper first proves that siRNA serves as

primers to transform the target mRNA into

dsRNA. The nascent dsRNA is degraded to

Eliminate the incorporated target mRNA

while generating new siRNAs in a cycle of

dsRNA synthesis and degradation.

Drosophila

GFP

Pp-Luc Photinus pyralis luciferase


Proved the nascent RNA is

double stranded..


AAAA-3’

5‘

Beyond upstream

siRNA’s antisense strand

The polarity of the RdRP reaction


Puzzlement

Puzzlement

How the ds siRNAs anneal to ss RNA?

Whether Target dsRNA can self replicate?

How to control the secondary RNAi?

How to explain the long-lasting of RNAi?



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