RNA Interferenz (RNAi) und ausgewählte Methoden der Genmanipulation

Transkript

1 RNA Interferenz (RNAi) und ausgewählte Methoden der Genmanipulation Prof. Dr. habil. Egon Amann 25. Januar 2012 Profilmodul: Grundlagen und Anwendungen der Genom- und Proteomforschung WS 2011/12 Frank Vitzthum und Egon Amann 1

2 Gliederung des Vortrags: Die Welt der RNA / Präbiotik The RNAi Revolution Wie funktioniert RNA Interferenz? Anwendungen der RNA Interferenz Ausgewählte Methoden der Genmanipulation 2

3 Gliederung des Vortrags: Die Welt der RNA / Präbiotik The RNAi Revolution Wie funktioniert RNA Interferenz? Anwendungen der RNA Interferenz Ausgewählte Methoden der Genmanipulation 3

4 Timeline of events pertaining to the early history of life on earth, with approximate dates in billions of years before the present. G.F. Joyce, Nature 418, 214 (2002) 4

5 Prebiotic clutter surrounding RNA Each of the four components of RNA (colored green, red, purple and blue) would have been accompanied by several closely related analogues (listed in in black). G.F. Joyce, Nature 418, 214 (2002) 5

6 Candidate precursors to RNA during the early history of life on earth a, Threose nucleic acid; b, peptide nucleic acid; c, glycerol-derived nucleic-acid analogue; d, pyranosyl-rna. B, nucleotide base G.F. Joyce, Nature 418, 214 (2002) 6

7 Hypothetical pathway for RNA-catalysed synthesis of RNA G.F. Joyce, Nature 418, 214 (2002) 7

8 Hypothetical pathway for RNA-catalysed protein synthesis Source: G.F. Joyce, Nature 418, 214 (2002) 8

9 Mechanism of RNA-catalysed self-cleavage a, General mechanism b, Secondary structure of the hammerhead ribozyme c, Crystal structure of the hammerhead ribozyme J.A. Doudna & T. Cech, Nature 418, 222 (2002) 9

10 Source: J.A. Doudna, T.R. Cech, Nature 418, 222 (2002) 10

11 RNA Nomenclature ssrna = single stranded RNAs dsrnas = double stranded RNAs utrnas = untranslated RNAs ncrnas (rrna, trna, introns, 5 and 3 untranslated regions, transposable elements, intergenic regions, micrornas) 97% of the transcriptional output is represented by ncrnas! snornas = small nucleolar RNAs tncrna = tiny non-coding RNAs sirnas = short interfering RNAs smrna = small modulatory RNAs 11

12 Menschliche RNA, kodiert im Zellkern W. Buselmaier, G. Tariverdian, Humangenetik f. Biologen,

13 Gliederung des Vortrags: Die Welt der RNA / Präbiotik The RNAi Revolution Wie funktioniert RNA Interferenz? Anwendungen der RNA Interferenz Ausgewählte Methoden der Genmanipulation 13

14 The Nobel Prize in Physiology or Medicine 2006 "for their discovery of RNA interference - gene silencing by double-stranded RNA Andrew Z. Fire Stanford University School of Medicine, Stanford, CA, USA b Craig C. Mello University of Massachusetts Medical School Worcester, MA, USA b

15 Why is the discovery of RNAi so important? RNAi is an evolutionary ancient method of genome defense in many organisms. RNAi is a remarkable RNA processing mechanism to underlie many distinct biological phenomena. RNAi is an important cellular method for gene expression regulation. RNAi is an extremely useful experimental tool for learning what genes do. RNAi is important in drug discovery and disease therapy (e.g., new drugs in fighting i.e. HIV, viral hepatitis, cancer). 15

16 RNAi as genome defense mechanism Short interfering RNAs may have a general role in silencing of transposable elements ( jumping genes ), repetitive genes (including transgenes) and viruses. About half of the sequences in the genome, for instance, were generated by duplication and insertion of transposons parasitic elements that create junk DNA. Silencing of these mobile elements is crucial for the stability of the genome, and RNAi-related processes involving the generation of short RNAs are essential to this silencing process in many organisms. Evidence 1: Worms with mutations in genes of the RNAi pathway are unable to silence transposons in germ line tissues. Evidence 2: Plants with mutations of the RNAi pathway show defects in the silencing of specific mobile genetic elements. 16

17 Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans Fire, A., S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, C.C. Mello SUMMARY Nature 391, (1998) Injecting dsrna into the worm was found to silence genes whose sequence were complementary to those of the introduced dsrna. dsrna was substantially more effective at producing interference than was either strand individually. The effect of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected dsrna were required per affected cell, suggesting that there could be a catalytic or amplification component in the interference process. 17

18 Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans Fire, A., S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, C.C. Mello Nature 391, (1998) EXPERIMENTS (I) The unc-22 gene encodes an abundant but nonessential myofilament protein. Several thousand copies of unc-22 mrna are present in each striated muscle cell. Decreases in unc-22 activity produce an increasingly severe twitching phenotype, whereas complete loss of function results in the additional appearance of muscle structural defects and impaired mobility. Purified antisense and sense RNAs covering a 742-nucleotide segment of unc-22 was used (using phagemid clones and T3 and T7 polymerases). The phenotype produced by interference using unc-22 dsrna was extremely specific. Progeny and injected animals exhibited behavior that precisely mimics loss-of-function mutations in unc

19 Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans Fire, A., S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, C.C. Mello Nature 391, (1998) EXPERIMENTS (II) A long interval (> 1 h) between sequential injections of sense and antisense RNA resulted in a dramatic decrease in interfering activity. dsrna produces a pronounced decrease or elimination of the endogenous mrna transcript. dsrna segments corresponding to various intron and promoter sequences did not produce detectable interference Results were confirmed with three additional genes with well characterized phenotypes: unc-54, fem-1, and hlh-1. To examine interference effects of dsrna at a cellular level, a transgenic line expressing two different green fluorescent protein (GFP)-derived fluorescent-reporter proteins in body muscle. Injection of dsrna directed gfp produced marked decrease in the fraction of fluorescent cells. 19

20 Effects of sense, antisense and mixed RNAs on progeny of injected animals A. Fire et al., Nature 391, 806 (1998) 20

21 Effects of mex-3 RNA interference on levels of the endogenous mrna a, Negative control showing lack of staining in the absence of the hybridization probe; b, Embryo form uninjected parent (showing normal pattern of endogenous mex-3 RNA; c, Embryo from parent injected with purified mex-3b antisense RNA; d, Embryo from parent injected with dsrna corresponding to mex- 3B. Source: A. Fire et al., Nature 391, 806 (1998) 21

22 RNA interference in C. elegans Silencing of GFP reporter in C. elegans occurs when animals feed on bacteria expressing GFP dsrna (a) but not in animals that are defective for RNAi (b). Note that silencing occurs through-out the body of the animals, with the exception of a few cells in the tail that express some residual GFP. The signal is lost in intestinal cells near the tail (arrowhead) as well as near the head (arrow). The lack of GFP-positive embryos in a (bracketed region) demonstrates the systemic spread and inheritance of silencing. Source: C.C. Mello, D. Conte, Nature 431, 338 (2004) 22

23 Double-stranded RNA can be introduced experimentally to silence target genes of interest a A silencing signal moves from veins into the leaves; b C. elegans left: control dsrna, right: GFP dsrna; c HeLa cells ORC6 sirna; d Drosophila expresses a hairpin to the white gene (left) 23

24 Tobacco plant infected with tobacco ringspot virus. The virus causing the initial symptoms had activated viral RNA silencing that inhibited spread of the infection into the upper leaves, and caused them to be specifically immune to tobacco ringspot virus secondary infection. D. Baulcombe, Nature 431, 356 (2004) 24

25 Zusätzliche Kopien des Gens Dihydroflavonol-Reduktase können die Produktion der Blütenfarbstoffe in Petunien verringern C. Napoli et al, Plant Cell 2, 279 (1990) 25

26 What are micrornas? (I) MicroRNAs are a group of small noncoding RNA (ncrna) molecules, distinct from but related to small interfering RNAs (sirnas), that have been identified in a variety of organisms. These small nucleotide (nt) RNAs are transcribed as part of longer molecules several kilobases in length that are processed in the nucleus into hairpin RNAs of nt by the dsrna-specific ribonuclease Drosha. The hairpin RNAs are transported to the cytoplasm, via an exportin 5-dependent mechanism, where they are digested by a second, dsrna-specific ribonuclease called Dicer. In animals, single-stranded microrna binds specific mrna through sequences that are significantly, though not completely, complementary to the target mrna, mainly to the 3 -untranslated region. 26

27 What are micrornas? (II) The bound mrna remains untranslated, resulting in reduced levels of the corresponding protein; alternatively, the bound mrna can be degraded, resulting in reduced levels of the corresponding transcript. Initially, it was estimated that there could be from 200 to 1000 microrna genes in the mammalian genome (1-3% of known genes are represented by micrornas). Today (2012) the number of micrornas is far over 1000 and still growing. The majority of micrornas (70%) are located in introns and/or exons, and 30% are located in intergenic regions. Three classes: Group 1: micrornas from introns and/or exons oriented in sense with the exon-coding host gene and, therefore, are transcribed as part of the annotated genes. 27

28 What are micrornas? (III) Group 2: micrornas are transcribed from intergenic regions or gene deserts, indicating that they form independent transcription units; Group 3: micrornas from introns and/or exons of annotated genes but are transcribed in the antisense orientation, suggesting that they too form their own transcription units. Tightly linked micrornas may be transcribed as polycistronic messengers; however, micrornas separated by more than 50 kb tend to represent independent transcription units. RNA polymerases II and III are candidates for primary transcription. Discovery: lin-4, a gene known to control the timing of C. elegans larval development, does not code for any protein but for a pair of small RNAs (shorter lin-4 > founding member). 28

29 Gliederung des Vortrags: Die Welt der RNA / Präbiotik The RNAi Revolution Wie funktioniert RNA Interferenz? Anwendungen der RNA Interferenz Ausgewählte Methoden der Genmanipulation 29

30 RNA silencing pathways in different organisms RNA Induced Silencing Complex G.J. Hannon, J.J. Rossi, Nature 431, 371 (2004) 30

31 Short interfering RNAs posttranscriptional gene silencing C.D. Novina, P.A. Sharp, Nature 430, 161 (2004) 31

32 MicroRNAs mechanism of translational silencing C.D. Novina, P.A. Sharp, Nature 430, 161 (2004) 32

33 Structure of the Giardia intestinalis ribonuclease Dicer I.J. MacRae et al., Science 311, 195 (2006) 33

34 Domain structure of representative members of proteins involved in RNAi or transcriptional gene silencing G. Meister, T. Tuschl, Nature 431, 343 (2004) 34

35 RNA silencing pathways in different organisms G. Meister, T. Tuschl, Nature 431, 343 (2004) 35

36 Dicer and RISC (RNA-induced silencing complex) G.J. Hannon, Nature 418, 244 (2992) 36

37 Small interfering RNAs versus small temporal RNAs G.J. Hannon, Nature 418, 244 (2992) 37

38 A model for the mechanism of RNAi G.J. Hannon, Nature 418, 244 (2992) 38

39 Model depicting distinct roles for dsrna in a network of interacting silencing pathways C.C. Mello, D. Conte, Nature 431, 338 (2004) 39

40 Main characteristics of short RNAs C.D. Novina, P.A. Sharp, Nature 430, 161 (2004) 40

41 Gliederung des Vortrags: Die Welt der RNA / Präbiotik The RNAi Revolution Wie funktioniert RNA Interferenz? Anwendungen der RNA Interferenz Ausgewählte Methoden der Genmanipulation 41

42 Genome-wide screens using RNAi G.J. Hannon, J.J. Rossi, Nature 431, 371 (2004) 42

43 Proposed scheme for the treatment of HIV patients using lentiviral vectors to transduce antihiv shrna into the patient s haematopoietic stem cells G.J. Hannon, J.J. Rossi, Nature 431, 371 (2004) 43

44 Gliederung des Vortrags: Die Welt der RNA / Präbiotik The RNAi Revolution Wie funktioniert RNA Interferenz? Anwendungen der RNA Interferenz Ausgewählte Methoden der Genmanipulation 44

45 Genveränderung (gene targeting) durch homologe Rekombination a Insertionsvektormethode > geringe Effizienz, häufig zufällige Integration (PCR!) b Austauschvektormethode > doppelte Rekombination, Genkonversion. Duales Selektionsverfahren auf G418- und Gancyclovir-resistente Zellen möglich. T. Strachan, A.P. Read, Molekulare Humangenetik,

46 Einführen genauer Mutationen durch gezielte Genveränderung Links: Hit-and-run -Methode wird mit Insertionsvektoren durchgeführt. Die punktgenaue Mutation liegt auf dem ersten Vektorkonstrukt. Durch eine Rekombination innerhalb des Chromosoms werden das Markergen und der Vektor entfernt. Rechts: Tag-and-exchange -Methode wird mit Austauschvektoren durchgeführt. Ein zweites Vektorkonstrukt wird dazu verwendet, die Mutation zu ersetzen, die im ersten Schritt eingeführt wurde. Das zweite Konstrukt enthält einen gegenselektierbaren Marker außerhalb des homologen Bereiches, um ein zufällige Integration auszuschließen. T. Strachan, A.P. Read, Molekulare Humangenetik,

47 Struktur der loxp-erkennungssequenz Die zentrale Sequenz mit acht Basenpaaren, die von einer umgekehrten Wiederholungssequenz mit 13 Basenpaaren flankiert wird, ist asymmetrisch und legt die Orientierung fest. T. Strachan, A.P. Read, Molekulare Humangenetik,

48 Geninaktivierung mit dem Cre-loxP-Rekombinationssystem T. Strachan, A.P. Read, Molekulare Humangenetik,