Conditional systems - principles

Conditional systems may function on the basis of:

- regulatory proteins

- aptamers

- allosteric ribozymes

- antisense RNA

- RNAi

RNA interference – The Beginning

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

mex-3 RNA

A control: not stained

B: wt

C: wt + antisense RNA

D: wt + ds 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 cellB) 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 silencingMET as a part of RISC

C.elegance: polycomb-dependent mechanism,polycomb proteins ass. with RISCchromatin remodeling: open – close transition

small-temporal RNAs

let7, lin4 negative regulator of genes70nt precurser, processed by DICER, results not in dsRNAbind target and prevent ribosomal elongation

RNA interference

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

The post-genomic era opens: Identification of the biological function !!!

Functional genomic screen to identify genes required for cell devision in C. elegans

Chromosom III: 2,300 predicted open reading frames96% inhibited by RNA-mediated interference

RNAi – Proof of Concept

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

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.

In vivo time-lapse differential interference contrast microscope

identification 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

RNAi as tool - companies

target identificationtarget validationlead compound screening

RNAi – manufacturing

RNAi – Literature

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 ....