RNA Interference
Graduate School of Biotechnology
and Biomedical Science
History
• Post-transcriptional gene silencing (PTGS) was observed first in Petunia
• dsRNA caused gene silencing in Caenorhabditis elegans –RNAi
• RNAi was also observed in Drosophila
Zhang H, Kolb FA, Jaskiewicz L, Westhof E, Filipowicz W.
Single processing center models for human Dicer and bacterial RNase III. Cell. 2004 Jul 9;118(1):57-68.
Dicer
Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference.Nature. 2001 Jan 18;409(6818):363-6.
A
Extracts prepared from for 0–12 h Drosophilaembryos or Drosophila S2 cells. Extracts were incubated for 0, 15, 30 or 60 min (left to right) with a uniformly labeled dsRNA. A 22-nucleotide marker prepared by in vitro transcription of a synthetic template is indicated (M).
A B
B
Drosophila S2 cells transfected with plasmids that direct expression of T7-epitope-tagged versions of Drosha,CG4792/Dicer-1 and Homeless or untagged -galactosidase.
Proteins were immunoprecipitated and incubated with cyclin E dsRNA for 0 or 60 min. Reactions in Drosophila embryo and S2 cell extracts are shown.
Dicer mediates the production of 21-23 nt RNA fragments upon incubation
of dsRNA in Drosophila embryo or S2 cell lysate
What are the substrates of Dicer?
dsRNA viruses
dsRNA resulting from
bidirectional transcription or RNA-dependent RNA synthesis
microRNA
(miRNA)
RdRP
Lee RC, Feinbaum RL, Ambros V.
The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993 Dec 3;75(5):843-54.
lin-4: the first identified miRNA
Predicted precursor structures of D. melanogaster miRNAs.
Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science. 2001 Oct 26;294(5543):853-8.
Transcriptional silencing
Inhibition of translation mRNA degradation
