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 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
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
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.
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.
Chromosom III: 2,300 predicted open reading frames
96% inhibited by RNA-mediated interference
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 identification
target validation
lead 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 ....