The mechanism of RNAi

The mechanism of RNAiThe mechanism of RNAi

by Renchangby 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 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

Dalmay, 2000Smardon, 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, Cogoni, et al, Gene silencing in Neurospora crassa reqiures Gene silencing in Neurospora crassa reqiures a protein homologous to RNA-dependent RNa protein homologous to RNA-dependent RNA polymerase, A polymerase, Nature 399, 166-169Nature 399, 166-169

QDE-1 N. crassaQDE-1 N. crassa

Smardon. A., et al, Smardon. A., et al, EGO-1 is related to RNA-directed RNA polymerase and EGO-1 is related to RNA-directed RNA polymerase and functions in gern-line development and RNA interferefunctions in gern-line development and RNA interference in C. elegans. nce in C. elegans. 2000, curr.Biol. 10, 169-178.2000, curr.Biol. 10, 169-178.

EGO-1, C. elegansEGO-1, C. elegans

Cell, Vol. 101, 543-553

Material: Arabidopsis, transgenesMaterial: Arabidopsis, transgenes

PVS:GFP: responsible for initiation of PTGSPVS:GFP: responsible for initiation of PTGSGFP: reporter of silencingGFP: reporter of silencingFour genetic loci are requried for PTGSFour genetic loci are requried for PTGSSde Sde SSilencing ilencing dedefectivefectiveSde1, Sde2, Sde3, Sde4Sde1, 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

GFPPp-Luc Photinus pyralis luciferase

Proved the nascent RNA is double stranded..

5‘ AAAA-3’

Beyond upstream siRNA’s antisense strand

The polarity of the RdRP reaction

PuzzlementPuzzlement

1.1. How the ds siRNAs anneal to ss RNA?How the ds siRNAs anneal to ss RNA?

2.2. Whether Target dsRNA can self replicate?Whether Target dsRNA can self replicate?

3.3. How to control the secondary RNAi?How to control the secondary RNAi?

4.4. How to explain the long-lasting of RNAi?How to explain the long-lasting of RNAi?

Good night