RNA-Regulation: RNA Interference
Literature
Martens: BIOspektrum 4/02 8. Jahrgang
M. Kuhlmann: Biol. Unserer Zeit Nr.3 (2004), S. 142.
Genregulation by
Transcription Factors
(Jacob&Monod)
RNA Interference
(Fire&Mello)
RNA Molecules can be suppressed, e.g.
double stranded RNA can trigger the decay
of RNA and thus silence the gene on a post
transcriptional level.
Nobel Price for Physiology/Medicine 2006
Andrew Z. Fire
and Craig C. Mello
for their discovery of
"RNA interference - gene silencing by double-stranded RNA"
The simplest of all viral life cycles.
The hypothetical virus shown
consists of a small double-stranded
DNA molecule that codes for only a
single viral capsid protein. No known
virus is this simple.
The life cycle of the Semliki forest virus.
The virus parasitizes the host cell for most of its biosyntheses.
Examples of viral Genomes
RNAtobacco mosaic virus
parvovirus
DNAT4 bacteriophage
fX174 bacteriophages
SV40
The life cycle of a retrovirus.
The enzyme reverse transcriptase first makes a DNA copy of the viral RNA molecule and
then a second DNA strand, generating a double-stranded DNA copy of the RNA genome.
The integration of this DNA double helix into the host chromosome, catalyzed by the
viral integrase, is required for the synthesis of new viral RNA molecules by the host-cell
RNA polymerase.
Transposons
Transfektion
Plasmid-DNA
genomic
DNA
mRNA
proteins
The introduction of foreign DNA
into the cell nucleus allows the
expression of arbitrary proteins.
Nucleic acid is a potentially powerful drug:
transient expression or stable transformation of forgein genes in human cells
genomic
DNA
mRNA
proteins
therapeutic
plasmid
Gene transfer into eucaryotic cells
mechanical methods
microinjection
electroporation
chemical vectors
Ca++ phosphate
cationic polymers
cationic liposomes
non-viral
gene delivery systems
viral vectors
retroviruses (Rv)
Adenovirus (Ad)
AAV, SV40
Kationic Liposomes are efficient
Transfektion Reagents
„lipoplexes“
lipid-DNA-complexes
DNA
DNA and Liposomes
fuse to a fluid crystalite aggregates
CATIONIC AMPHIPHILES
+
+
DOTAP
O
CH3
H3C N
H
CH3
+
+
+
+
O
O
Silvius, 1986
O
+
+
+
+
+
+
DDAB
+
Cationic Liposome
H3C
N
H3C
Pinnuduwage, B.B.A. ,1989
NH3
DOGS
NH2
O
C N
H2N
H3N
C
O
Behr, PNAS ,1989
Felgner et al. PNAS 1987
Gene Delivery Mediated by Synthetic Reagents
cationic
liposomes
- Transfer across many barriers
k0
DNA
complex formation
k1
endocytosis
k2
endosomal breakup
Golgi
DNase
ER
k3
nuclear translocation
nucleus
Monitoring Gene Expression via Reporter Genes
Green Fluorescent Protein GFP
optical real time assay
firefly enzyme:
GFP expressing cell culture
Luciferase as reporter : capable of
emitting light through ATP, O2
dependent oxidationof luciferin
bacterial enzymes:
e.g.
b-Galactosidase
Chloramphenicol-Acetyl-Transferase
The Antisense Strategy
By adding Antisense RNA specific mRNA
molecules are blocked for translation
The Experiment of Fire und Mello
RNA carrying the code for a muscle protein is injected into the worm C.
elegans. Single-stranded RNA has no effect. But when double-stranded RNA
is injected, the worm starts twitching in a similar way to worms carrying a
defective gene for the muscle protein.
RNA silencing
1. DICER: analog to
RNase III
2. siRNA
(small interfering RNA)
3. RISC: RNAi-inducing silencing complex
(with unknown subunit: SLICER)
Standard model of RNAi:
RISC:
RNAi-induced
silencing complex
double stranded RNA is cut by Dicer (a homolog of the dsRNA-specific
RNase III) into siRNAs, siRNAs are bound by RISC and unwound, the
antisense strand specifies RISC (RNA Induced Silencing complex mit
ssRNase-Aktivität) to degrade the target mRNA.
Alternative RNAi Mechanism
Unwound siRNAs are used by RNA
dependent RNA-Polymerase RdRP as
Primer to replicate mRNA into a new
double strand. As this is again the
substrate for Dicer, degradation of
mRNA by RISC is theoretically not
necessary.
Connection between Antisense RNA and RNAi
Natural function on Gene Silencings
Cellular function of RNAi
Degradation of aberrant RNAs and RNA pieces
Post-transkriptional Genregulation by endogene
Antisense-RNAs
Suppression of translocating genes (Transposons)
Preservation of chromosomal integrity by RNA-directed
DNA Methylierung
Defense against Retrovirus (z.B. Aids, TMV etc.)
Retro-Virus indiced Gene Silencing
Molecular Sisters:
siRNA and miRNA
endogeneously coded microRNA (miRNA) is cut into
pieces by the RNAi
Mechanism, which can
supress translation
specifically.
Gene „knock down“
siRNA is brought into isolated cells
(transfection) and the mRNA of the target
genes is degraded. The resulting reduction
of gene product (knock down) allows it to
determine the physiological function of the
target gene.
genomic
DNA
mRNA
RISC
proteins
siRNA
Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001). "Duplexes of 21-nucleotide RNAs
mediate RNA interference in cultured mammalian cells". Nature 411 (6836): 494-8.
Example : siRNA to target HIV
Strategy for siRNA against HIV: Attack of RNA immediately after intrusion
of the RNA into the cells, and before the Reverse Transkriptase can
transform the viral RNA into cDNA.
siRNA Therapy against Cancer
An approach is to use siRNAto suppress mutated p53-Proteins. Wildtyp-p53 acts as
Tumorsuppressor by forcing the cell into apoptosis or cell cycle arrest. Sporadic mutations
in one of the allele of the p53-Gen act dominantly and hinders apoptosis or cell cycle arrest.
Inhibition by siRNA of the mutated p53-Allels could stop the development of cancer.
Conclusion
RNAi ...
* Can trigger the degradation of mRNA
* Needs "Dicer“, „RISC“ und RdRP
* Can known down specific mRNA (and thus genes)
* Is part of the "old" molecular immune system to control RNA
* Can be used at two levels, both in therapy and in research.