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RNA-Regulation: RNA Interference

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Title: Raten-Theorie chemischer Reaktionen Author: Joachim R dler Last modified by: Braun Lab Created Date: 5/1/2005 2:18:08 PM Document presentation format – PowerPoint PPT presentation

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Title: RNA-Regulation: RNA Interference


1
RNA-Regulation RNA Interference
Literature Martens BIOspektrum 4/02 8.
Jahrgang M. Kuhlmann Biol. Unserer Zeit Nr.3
(2004), S. 142.
2
(No Transcript)
3
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.
4
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"
5
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.
6
The life cycle of the Semliki forest virus. The
virus parasitizes the host cell for most of its
biosyntheses.
7
Examples of viral Genomes
RNAtobacco mosaic virus
parvovirus
SV40
DNAT4 bacteriophage
fX174 bacteriophages
8
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.
9
Transposons
10
Transfektion
Plasmid-DNA
genomic DNA
mRNA
The introduction of foreign DNA into the cell
nucleus allows the expression of arbitrary
proteins.
proteins
11
Nucleic acid is a potentially powerful drug
transient expression or stable transformation of
forgein genes in human cells
therapeutic plasmid
12
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
13
Kationic Liposomes are efficient Transfektion
Reagents
lipoplexes lipid-DNA-complexes
DNA
DNA and Liposomes fuse to a fluid crystalite
aggregates
14
CATIONIC AMPHIPHILES



DOTAP






Silvius, 1986




DDAB
Cationic Liposome
Pinnuduwage, B.B.A. ,1989
DOGS
Behr, PNAS ,1989
Felgner et al. PNAS 1987
15
Gene Delivery Mediated by Synthetic Reagents
- Transfer across many barriers
k0
complex formation
k1
endocytosis
k2
endosomal breakup
k3
nuclear translocation
16
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
17
The Antisense Strategy
  • By adding Antisense RNA specific mRNA molecules
    are blocked for translation

18
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.
19
RNA silencing
  1. DICER analog toRNase III
  2. siRNA(small interfering RNA)

20
3. RISC RNAi-inducing silencing complex (with
unknown subunit SLICER)
21
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.
22
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.
23
Connection between Antisense RNA and RNAi
24
Natural function on Gene Silencings
25
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.)
26
Retro-Virus indiced Gene Silencing
27
Molecular SisterssiRNA and miRNA
endogeneously coded micro-RNA (miRNA) is cut into
pieces by the RNAi Mechanism, which can supress
translation specifically.
28
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.
29
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.
30
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.

31
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.
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