Conditional systems - principles Conditional systems may function on the basis of: - regulatory proteins - aptamers - allosteric ribozymes - antisense RNA - RNAi
RNA interference – The Beginning mex-3 RNA A control: not stained B: wt C: wt + antisense RNA D: wt + ds RNA Fire et al. '98 "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans " Nature 391: 806-11 Introduction of RNA into cells to interfere with function of an endogeneous gene Investigation of the requirements for structure and delivery of interference RNA !!! ds mixture causes potent and specific interference !!!! !!! ds RNA substancially more effective than antisence !!! !!! effect were evident in both the injected animals and their progeny !!!
RNA interference “RNA interference (RNAi) represents an evolutionary conserved cellular defense mechanism for controlling the expression of alien genes in filamentous fungi, plants, and animals. It is caused by sequence-specific mRNA degradation, and is mediated by double-stranded RNA (dsRNA) homologous in sequence to the target RNA.” Defense mechanism dsRNA is often a byproduct of viral replication or is formed by aberrant transcription from genetic elements after random integration in the host genome.
RNA interference - Mechanism DICER - RNAse III, ds spec. endonuclease - Dimer, 2 catal. domains, helicase and PAZ motif - produce 2-3nt 3´overhangs - ATP-dependent ribonuclease RISC - RNA-induced silencing complex - RISC contains siRNA - precurser activated by ATP - find and destroy mRNA of complementary sequence - contains endo- and exonuclease, cleaves the hybrid in the middle imm. followed by degradation - ARO: PAZ domain (assembly)
Amplification and Spreading of Silencing RNAi spread throughout the organism requirement: A) pass from cell to cell B) amplification of the signal A) SID required for silencing, transmembrane protein (may be channel for import) B) RdRP - RNA-dep-RNA polymerase - in some organisms (drosophila, plants) - concentrate si RNA by amplification - siRNA might prime the synthese of additional ds siRNA
Transcriptional Gene Silencing plant: methylation in promotor regions leads to gene silencing MET as a part of RISC C.elegance: polycomb-dependent mechanism, polycomb proteins ass. with RISC chromatin remodeling: open – close transition
small-temporal RNAs let7, lin4 negative regulator of genes 70nt precurser, processed by DICER, results not in dsRNA bind target and prevent ribosomal elongation
RNAi for analysis of gene function and as therapeutic - duplexes of 21-nt small interfering RNAs (siRNAs) - guide sequence-specific degradation of the homologous mRNA degradation of targeted mRNAs, "knock-down" targeting of essential genes causes growth arrest or triggers apoptosis
RNAi - Advantages - dsRNA is the interfering agent (stability) it is highly specific it is remarkably potent (only a few dsRNA molecules per cell are required for effective interference) the interfering activity can cause interference in cells and tissues far removed from the site of introduction
RNAi – Proof of Concept The post-genomic era opens: Identification of the biological function !!!Functional genomic screen to identify genes required for cell devision in C. elegansChromosom III: 2,300 predicted open reading frames96% inhibited by RNA-mediated interference Gönczy et al.: Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature. 2000 408(6810):331-6.
RNAi – cell devision in C. elegans In vivo time-lapse differential interference contrast microscopeidentification of 133 genes (6%) necess. for distinct cellular processes in early embryos (most of the genes of CIII that are required for proper cell devision)47% of the identified genes have ortholoques in other eukaryotes Gönczy et al.: Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature. 2000 408(6810):331-6.
target identification target validation lead compound screening RNAi as tool - companies
1.Tuschl T. Expanding small RNA interference. Nat Biotechnol (2002); Vol. 20(5): pp. 446-8. 2.Hammond S.M., Boettcher S., et. al. Argonaute2, a Link Between Genetic and Biochemical Analyses of RNAi. Science (2001); Vol. 293: pp. 1146-50. 3.Zamore P.D. Ancient Pathways Programmed by Small RNAs. Science (2002); Vol. 296: pp. 1265-1269. 4.Tabara H., Sarkissian M., et. al. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell (1999); Vol. 99(2): pp. 123-32 5.Lee N.S., Dohjima T., et. al. Expression of Small Interfering RNAs Targeted Against HIV-1 Rev Transcripts in Human Cells. Nat Biotechnol (2002); Vol. 20(5): pp. 500-5. and, and, and .... RNAi – Literature