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. Genregulation byTranscription Factors (Jacob&Monod)

3. 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.

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. genomic DNA mRNA proteins 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-Transferase

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 DICER: analog toRNase III 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 Sisters:siRNA 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.