RNA-Regulation: RNA Interference

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RNA-Regulation RNA Interference
Literature Martens BIOspektrum 4/02 8.
Jahrgang M. Kuhlmann Biol. Unserer Zeit Nr.3
(2004), S. 142.
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(No Transcript)
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RNA Interference
(FireMello)
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.
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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"
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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.
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The life cycle of the Semliki forest virus. The
virus parasitizes the host cell for most of its
biosyntheses.
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Examples of viral Genomes
RNAtobacco mosaic virus
parvovirus
SV40
DNAT4 bacteriophage
fX174 bacteriophages
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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.
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Transposons
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Transfektion
Plasmid-DNA
genomic DNA
mRNA
The introduction of foreign DNA into the cell
nucleus allows the expression of arbitrary
proteins.
proteins
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Nucleic acid is a potentially powerful drug
transient expression or stable transformation of
forgein genes in human cells
therapeutic plasmid
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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
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Kationic Liposomes are efficient Transfektion
Reagents
lipoplexes lipid-DNA-complexes
DNA
DNA and Liposomes fuse to a fluid crystalite
aggregates
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CATIONIC AMPHIPHILES



DOTAP






Silvius, 1986




DDAB
Cationic Liposome
Pinnuduwage, B.B.A. ,1989
DOGS
Behr, PNAS ,1989
Felgner et al. PNAS 1987
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Gene Delivery Mediated by Synthetic Reagents
- Transfer across many barriers
k0
complex formation
k1
endocytosis
k2
endosomal breakup
k3
nuclear translocation
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Monitoring Gene Expression via Reporter Genes
Green Fluorescent Protein GFP optical real time
assay
firefly enzyme
Luciferase as reporter capable of emitting
light through ATP, O2 dependent oxidationof
luciferin
GFP expressing cell culture
bacterial enzymes
e.g. b-Galactosidase Chloramphenicol-Acetyl-Transf
erase
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The Antisense Strategy

• By adding Antisense RNA specific mRNA molecules
  are blocked for translation

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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.
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RNA silencing

1. DICER analog toRNase III
2. siRNA(small interfering RNA)

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3. RISC RNAi-inducing silencing complex (with
unknown subunit SLICER)
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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.
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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.
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Connection between Antisense RNA and RNAi
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Natural function on Gene Silencings
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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.)
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Retro-Virus indiced Gene Silencing
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Molecular SisterssiRNA and miRNA
endogeneously coded micro-RNA (miRNA) is cut into
pieces by the RNAi Mechanism, which can supress
translation specifically.
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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.
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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.
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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.

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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.