the mechanism of rnai
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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The mechanism of RNAiThe mechanism of RNAi
by Renchangby Renchang
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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
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Fire Andrew, et al, Potent and specific genetic interference by double-strand RNA in Caenorhabditis elegans. Nature, 1998, 391:806-811
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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.
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Bernstein, 2001
Hamilton, 1999
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Cogoni, 1999
Dalmay, 2000Smardon, 2000
Lipardi, 2001
Nykanen, 2001
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Sijen, 2001
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Hamilton, A.J., et al, A species of small antisense RNA in posttranscriptional gene silencing in plants. 1999, Science, 286, 950-952.
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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.
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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
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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
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Cell, Vol. 101, 543-553
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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
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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
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Proved the nascent RNA is double stranded..
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5‘ AAAA-3’
Beyond upstream siRNA’s antisense strand
The polarity of the RdRP reaction
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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?
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Good night