RNA-Regulation: RNA Interference

Literature

Martens: BIOspektrum 4/02 8. Jahrgang

M. Kuhlmann: Biol. Unserer Zeit Nr.3 (2004), S. 142.

Genregulation byTranscription 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

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.

Transposons

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

+

+

+

++ +

+

+

+

+++

+

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 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 toRNase III

2. siRNA(small interfering RNA)

3. RISC: RNAi-inducing silencing complex (with unknown subunit: SLICER)

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.

Connection between Antisense RNA and RNAi

Natural function on Gene Silencings

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.)

Retro-Virus indiced Gene Silencing

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.

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.