RNA-Regulation: RNA Interference
Literature
Martens: BIOspektrum 4/02 8. Jahrgang
M. Kuhlmann: Biol. Unserer Zeit Nr.3 (2004), S. 142.
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
SV40
fX174 bacteriophages
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.
The life cycle of a retrovirus.
Plasmid-DNA
genomic DNA
mRNA
proteins
Transfektion
The introduction of foreign DNA into the cell nucleus allows the expression of arbitrary proteins.
therapeutic plasmid
genomic DNA
mRNA
proteins
Nucleic acid is a potentially powerful drug:
transient expression or stable transformation of forgein genes in human cells
electroporation
chemical vectorsCa++ phosphate cationic polymerscationic liposomes
microinjection
mechanical methods
viral vectorsretroviruses (Rv)Adenovirus (Ad) AAV, SV40
Gene transfer into eucaryotic cells
non-viralgene delivery systems
cationicliposomes
DNS
DNA and Liposomesfuse to a fluid crystalite aggregates
DNA
„lipoplexes“lipid-DNA-complexes
Kationic Liposomes are efficient Transfektion Reagents
CH3
NH3C
CH3
O
HO
O
O
NH3C
H3C
NCO
CO
NH2
H2N
H3N
NH3
DOTAP
DDAB
DOGS
Silvius, 1986
Pinnuduwage, B.B.A. ,1989
Behr, PNAS ,1989
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+
+
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Cationic Liposome
CATIONIC AMPHIPHILES
Felgner et al. PNAS 1987
DNA
cationicliposomes
nucleus
Golgi
ER
DNase
complex formation
endocytosis
endosomal breakup
nuclear translocation
k1
k2
k3
k0
Gene Delivery Mediated by Synthetic Reagents
- Transfer across many barriers
Luciferase as reporter : capable of emitting light through ATP, O2 dependent oxidationof luciferin
Green Fluorescent Protein GFPoptical real time assay
e.g.-GalactosidaseChloramphenicol-Acetyl-Transferase
GFP expressing cell culture
bacterial enzymes:
firefly enzyme:
Monitoring Gene Expression via Reporter Genes
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.
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.
Standard model of RNAi:
RISC: RNAi-induced silencing complex
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.
Cellular function of RNAi
Post-transkriptional Genregulation by endogene Antisense-RNAs
Degradation of aberrant RNAs and RNA pieces
Suppression of translocating genes (Transposons)
Preservation of chromosomal integrity by RNA-directed DNA Methylierung
Defense against Retrovirus (z.B. Aids, TMV etc.)
Molecular Sisters:siRNA and miRNA
endogeneously coded micro-RNA (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.
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.
genomic DNA
mRNA
proteins
RISC
siRNA
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.