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Part I: Ribozymes

Prof. F.Allain 2/2/2004. Part I: Ribozymes. Part II: SELEX (RNA in-vitro evolution). Part III: RNAi RNA inhibition and silencing. The RNA world hypothesis. Part I: Ribozymes. A brief History. How many ribozyme ? Why ?. Catalytic efficiency, condition. 3D structure of ribozyme:

JasminFlorian
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Part I: Ribozymes

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  1. Prof. F.Allain 2/2/2004 Part I: Ribozymes Part II: SELEX (RNA in-vitro evolution) Part III: RNAi RNA inhibition and silencing

  2. The RNA world hypothesis

  3. Part I: Ribozymes A brief History How many ribozyme ? Why ? Catalytic efficiency, condition 3D structure of ribozyme: And a mechanism of catalysis Biological application ?

  4. A brief History 1982:Self-splicing in Tetrahymena pre-rRNA (group I intron) Kruger et al, and Cech, Cell 31, 147-157 (1982) 1983:RNAse P is a ribozyme Guerrier-Takada et al, and Altman, Cell, 35, 849-857 (1983)

  5. How many ribozyme ? Why ? - the hammerhead ribozyme (plant virus) - the hairpin ribozyme (plan virus) - hepatitis delta ribozyme (human virus) - neurospora VS ribozyme (mitochondrial RNA) - group I and group II intron ribozyme (rRNA and mt RNA) - RNAse P (tRNA maturation) - Ribosome (translation) - Spliceosome ?? (splicing)

  6. One main reaction: Nucleolytic cleavage Transesterification (SN2) Hammerhead Haipin Hepatitis delta VS ribozyme From Lilley TIBS (2003)

  7. The hammerhead ribozyme (plant virus) - discovered in small RNA satellites of small viruses (1986) - replication by rolling circle mechanism Secondary structure

  8. The hammerhead ribozyme (plant virus) - tertiary structure Scott et al and Klug, Science 1996

  9. The hairpin ribozyme (plant virus) From Lilley TIBS (2003)

  10. The hepatitis delta ribozyme (human virus) From Lilley TIBS (2003)

  11. Group I &IIintron ribozyme (rRNA and mt RNA) Doudna and Cech Nature, 2002

  12. Group Iintron ribozyme (rRNA and mt RNA) Golden et al, and cech Science (1998)

  13. Catalytic efficiency, condition - ribozyme follows a Michaelis-Menten kinetics k1 k2 E + S ES E + P k-1 k-1+k2 Km= kcat= 0.5-2 min-1 = 10-5-10-7 M k1 kcat/ Km= 103-106 M-1.min-1 Good catalytic efficiency!! - all ribozyme need cations for activity (Mg2+ ,Mn2+)

  14. 3D structure of ribozyme: mechanism of catalysis hairpin ribozyme Hepatitis delta ribozyme Ferre d’Amare, Nature 1998 Ruppert et al, Nature 2001, Science 2002

  15. How to catalyse the reaction ? From Lilley TIBS (2003)

  16. Structure of the hairpin ribozyme hairpin ribozyme Ruppert et al, Nature 2001, Science 2002

  17. hairpin ribozyme Ground state Transition state Ruppert et al, Nature 2001 Ruppert et al, Science 2002

  18. hairpin ribozyme free bound free bound Loop A Loop B

  19. hairpin ribozyme Transition state Ruppert et al, Science 2002

  20. Acid-Base catalysis ? (textbook Voet and Voet) like with RNAse A

  21. Acid-Base catalysis ? G8 as a base A38 as an acid Bevilacqua, Biochemistry 2003

  22. Hepatitis delta ribozyme Ferre d’Amare, Nature 1998

  23. Biological application ? Tentative of gene therapy with the hairpin and the hammerhead ribozyme against viral RNA for example.

  24. Reference: Reviews: Lilley TIBS (2003) De Rose Chem & Biol (2002) Ferre d’Amare Biopolymer (2003) Article: Kruger et al, and Cech, Cell (1982) Guerrier-Takada et al, and Altman, Cell (1983) Scott et al Nature (1995) Science (1996) Rupert et al Nature (2001), Science (2002)

  25. Part II: SELEX A brief History The method ? A few examples. Biological application ?

  26. SELEX :Systematic Evolution of Ligands by EXponential enrichment A brief History Ellington and Szostak, Nature (1990) Tuerk and Gold , Science (1990)

  27. In vitro selection of RNA molecules that bind specific ligands • Andrew D. Ellington & Jack W. Szostak • Subpopulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules. Roughly one in 1010 random sequence RNA molecules folds in such a way as to create a specific binding site for small ligands. • Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.Tuerk C, Gold L.High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.

  28. Wilson and Szostak, Ann.Rev.Bioc. (1999)

  29. - Selection against small molecules - Selection against proteins - Selection of new ribozymes (RNA world)

  30. The ATP aptamer structure

  31. C G C C C C C G C A G C C A U A C C G G/A U/G C U A Ny Nx C A G-C A-U G-C G-C U-G A• A•U G•A G-C G-C A N-N N-N N-N N-N G U G C U C-G G-C U-A A-U 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Consensus NRE selex NRE 5-20nM B1 10-50nM (5’ETS) Mouse B2 50-100nM (5’ETS) Mouse Nucleolin RNA Targets

  32. RBD2-RNA-RBD1“sandwich” RBD1 RBD2 F56 linker G16 22 3’ 3’ 1 5’ 5’ Allain et al, EMBOJ (2000)

  33. In vitro selection of an enzyme

  34. Reference: Reviews: Wilson and Szostak Ann.Rev.Bioch.(1999) Gold et al, Ann.Rev.Bioch.(1995)

  35. Part III: Introduction to RNAi A brief History RNAi Mechanism SiRNA and miRNA A few very recent structures Biological application A practical example of siRNA

  36. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans ANDREW FIRE*, SIQUN XU*, MARY K. MONTGOMERY*, STEVEN A. KOSTAS*†, SAMUEL E. DRIVER‡ & CRAIG C. MELLO‡ Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene,. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression,. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny.Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process. Nature V391 pp 806-811 (1998)

  37. + DS RNA against GFP Fire at al, Nature V391 pp 806-811 (1998)

  38. In situ mRNA hybridization of Mex3 RNA in Embryo -C +C with DS RNA From animal: with AntisensRNA Fire at al, Nature V391 pp 806-811 (1998)

  39. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.Zamore PD, Tuschl T, Sharp PA, Bartel DP. • Double-stranded RNA (dsRNA) directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Using a recently developed Drosophila in vitro system, we examined the molecular mechanism underlying RNAi. We find that RNAi is ATP dependent yet uncoupled from mRNA translation. During the RNAi reaction, both strands of the dsRNA are processed to RNA segments 21-23 nucleotides in length. Processing of the dsRNA to the small RNA fragments does not require the targeted mRNA. The mRNA is cleaved only within the region of identity with the dsRNA. Cleavage occurs at sites 21-23 nucleotides apart, the same interval observed for the dsRNA itself, suggesting that the 21-23 nucleotide fragments from the dsRNA are guiding mRNA cleavage • Cell, v101 pp25-33 (2000)

  40. dsRNAi is cut in 21-23 nt fragments • Zamore et al, Cell, v101 pp25-33 (2000)

  41. The mRNA is cut in 21-23 nt fragments by the siRNA • Zamore et al, Cell, v101 pp25-33 (2000)

  42. A first model for the mechanism RNAi • Zamore et al, Cell, v101 pp25-33 (2000)

  43. Role for a bidentate ribonuclease in the initiation step of RNA interference.Bernstein E, Caudy AA, Hammond SM, Hannon GJ..RNA interference (RNAi) is the mechanism through which double-stranded RNAs silence cognate genes. In plants, this can occur at both the transcriptional and the post-transcriptional levels; however, in animals, only post-transcriptional RNAi has been reported to date. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC, to destroy specific messenger RNAs. Here we identify an enzyme, Dicer, which can produce putative guide RNAs. Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain and dual RNase III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi. • Nature v 409, pp 363-366 (2001)

  44. Identification of DICER 22 nt RNA • Bernstein et al, Nature v 409, pp 363-366 (2001)

  45. The RISC complex Elbashir et al, G&D, v18 pp188-200 (2001)

  46. MicroRNAs. Genomics, Biogenesis, Mechanism, and Function.Bartel DP. • MicroRNAs (miRNAs) are endogenous approximately 22 nt RNAs that can play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. Although they escaped notice until relatively recently, miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes. • Cell, v116, pp 281-297 (2004) (review) SiRNA and miRNA

  47. First miRNA in C.elegans miRNA in Plants miRNA in C.elegans with homologs In flies and human

  48. A high number: about 1% of the genes Human: 200-255 miRNA C.elegans: 103-120 miRNA Drosophila: 96-124 miRNA

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