RNA Silencing, mechanism and applications

Transcription

1 RA Silencing, mechanism and applications

2 The Central Dogma of Molecular Biology

3 There is life outside proteincoding genes There are approximately gene predictions for the Human genome But the estimated number of protein coding genes is 21,561 It is estimated that 98% of the transcriptional output of the human genome represents RA that does not encode protein

4 RA Silencing, mechanism and applications 1. History and definitions 2. The role of small RAs in gene regulation 3. Mechanisms of RA interference 4. Biological role of RA silencing 5. Applications of RA silencing 6. miras

5 Posttranscriptional gene silencing (PTGS) Classical key experiment in petunia: Key enzyme for flower pigmentation: chalcone synthase (CHS) Artificially introduced 2 nd CHS gene Expected: more intense flowers Real outcome: Variable phenotypes, including white flowers

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8 Effects of expression of CHS sense and antisense RA on flower pigmentation in Petunia van der Krol et al., Plant Cell 2:291 (1990) + Plant Mol Biol 14:457 (1990).

9 CO-SUPPRESSIO The silencing of an endogenous gene due to the presence of a homologous transgene or virus. Co-suppression can occur at the transcriptional or posttranscriptional level.

10 Key experiment on RA interference (RAi) Injection of of ds dsra to to C. C. elegans causes gene-specific silencing ature 391, (1998); ew term: RA interference (RAi)

11 umber of Publications Entrez PubMed Search Terms: RAi or sira or mira

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13 RA silencing RA mediated impairment of transcription- TGS -methylation (shras, rasiras and siras?) RA mediated impairment of translation -PTGS (siras and some miras) and -translational arrest (miras)

14 Small RA classification small RAs Sequence specific RA degradation Inhibition of translation RA mediated methylation Various types of suppression Post- Transcriptinal gene silencing Gene regulation Regulation of the Epigenome Suppression of transposable elements stress response etc Short interfering RA (sira) microra mira rasiras, endosiras etc. pira tasira natsira

15 RA σίγηση, ένας μηχανισμός ρύθμισης και άμυνας 1. History 2. The role of small RAs in gene regulation

16 short interfering RA (sira) ca 21 ucleotides

17 short interfering RA (sira) 5 terminal phosphates 5 P ca 21 ucleotides -P two 3 protruding unpaired nucleotides - -

18 PTGS / RAi induction RA quant ( threshold ) aberrant RA Ectopic pairing? RdRP?? RdRP ds RA Target RA antisense RA

19 Basic Model for the Mechanism Rase III-type enzyme Dicer Target RA antisense RA

20 Basic Model Rase III-type enzyme Dicer Target RA antisense RA

21 Basic Model short interfering RA ( sira )

22 short interfering RA siras play an essential role in RA interference They are mediators as well as indicators of posttranscriptional gene silencing (PTGS)

23 Integration of sira to RISC Integration of sira to RISC - P 5 P - 5 ca 21 ucleotides Incorporation in RA-induced silencing complex (RISC complex)

24 Degradation of Target RA target RAs cleaved target RAs

25 Degradation of target RA: major players Dicer RISC sira

26 Dicer Dicer generates RAs with 2 nt 3 overhang and 5 phosphorylated terminus, both required for activity Fly Dicer requires ATP, human may not

27 EZYME STRUCTURE Rase III Bact.Rase III Class II: Drosha Class III dicer

28 Different organisms have different numbers of Dicers

29 Mello CC, Conte D; ature 431:338, 2004

30 Transgenic GFP expressing.benthamiana is our system

31 Silencing in plants is a non-cell autonomous event

32 The Mechanism of RA silencing is to a certain extent conserved between eucaryots

33 Testing plant extracts for their silencing potential mra GFP non-silenced si RA GFP silenced GFP-expressing RA extract C.elegans RA extract

34 RA silencing pathways in fungi

35 RA silencing pathways in fungi AS, ascomycete; BS, basidiomycetes; ZY, zygomycete; OO, oomycete; AC, Aspergillus clavatus; AFl, Aspergillus flavus; AFu, Aspergillus fumigatus; A, Aspergillus nidulans; AO, Aspergillus oryzae; A, Aspergillus terreus; CA, Candida albicans; CC, Coprinus cinereus; CL, Candida lusitaniae; C, Cryptococcus neoformans; CT, Candida tropicalis; FG, Fusarium graminearum; FO, Fusarium oxysporum; FV, Fusarium verticillioides; HC, Histoplasma capsulatum; MO, Magnaporthe oryzae; C, eurospora crassa; PC, Phanerochaete chrysosporium; PG, Puccinia graminis; PI, Phytophthora infestans; RO, Rhizopus oryzae; SC, Saccharomyces cerevisiae; S, Stagonospora nodorum; SP, Schizosaccharomyces pombe; UM, Ustilago maydis; AT, Arabidopsis thaliana.

36 In fungi there seems to be an avoidance of RAi when killer virus is present

37 RA σίγηση, ένας μηχανισμός ρύθμισης και άμυνας 1. History and definitions 2. The role of small RAs in gene regulation 3. Mechanisms of RA interference 4. Biological role of RA silencing

38 Biological role of silencing Protection against invasive RAs especially RA viruses Silencing of transposable elements Regulation of expression of endogenous genes

39 RA σίγηση, ένας μηχανισμός ρύθμισης και άμυνας 1. History 2. The role of small RAs in gene regulation 3. Mechanisms of RA interference 4. Biological role of RA silencing 5. Applications of RA silencing

40 Applications of RA silencing Functional analysis of genes Sequence specific suppression of unwanted genes Generation of plants resistant to viruses

41 Functional analysis of gene by reverse genetics 1. Identification of gene sequence 2. Reverse genetics: -find copy number/possibly locus (Arabidopsis) -charachterise time and space of expression -find gene function a. overexpression b. suppression (knock-down, knock-out)

42 RAi induced at the level of siras Direct application of sira (a) chemically synthesized (b) enzymatically synthesized Vector-mediated RAi (DA or virus) delivery via short hairpin RAs (shra)

43 Strategies of expressing double-stranded RA Promoter spacer/ intron terminator AAA Pol II and Pol III (type III) e.g U6 prom alternative: tandem promoters, opposing promoters

44 Hairpin induced gene silencing of CP gene 35S spacer ter nptii RB (+) CP frag. (-)CP frag. LB Dicer~ ~ ~ ~ RISC ~ ~ ~ ~

45 RA σίγηση, ένας μηχανισμός ρύθμισης και άμυνας 1. History and definitions 2. The role of small RAs in gene regulation 3. Mechanisms of RA interference 4. Biological role of RA silencing 5. Applications of RA silencing 6. miras

46 MicroRAs short (20-25nt) RA molecules translated as precursor RA molecule post transcriptional gene regulation target mra for cleavage or translational repression very abundant class of genes

47 The processing pathway of sira & mira from Dykxhoorn et al., (2003) ature Reviews 4,

48 Mechanism of mira suppression of gene expression Transcription mra degradation Translational repression 1 Initiation 2 Elongation 3 Termination 4 Release Co-translational degradation of the nascent peptide

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50 Cullen ature Immunology 7:

51 Identification of Argonaute 2 as Slicer in humans Published by AAAS J. Liu et al., Science 305, (2004)

52 mira The mira are endogenous small RA guides that repress the expression of target genes. Differ from sira in biogenesis not in functions, although mechanisms can be different. mra cleavage when complementarity is extensive, repress translation when not. lin-4 mutant worms had defects in timing of cell division. Encodes a small RA that binds to and silenced lin-14 message. Lin-14 mra levels do not decline, but that may not always be the case. let-7 also found in other species.

53 mira Many mira are embedded in introns of protein encoding genes and are transcribed together with host genes. mira can be expressed in developmentally tissue specific fashion but may not be expressed in tissues where putative target sequences are.

54 The structure of human pri-miras Du, T. et al. Development 2005;132:

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56 Useful plant mira properties (at least some) miras are conserved between species most precursor sequences are not conserved precursor structure is somewhat conserved mature mira always cut from 1 arm of RA helix in precursor plant precursors are more complex

57 Plasterk Cell 124:877, 2006

58 PLoS, 39 (3):

59 Summary of plant mira/target recognition 1. Perfect match for positions o more than 2 consecutive mismatches at 3 3. Free energy at least 72% that of perfect match

60 MicroRAs can function as tumour suppressors and oncogenes Esquela-Kerscher et al. ature Reviews Cancer 6, (April 2006) doi: /nrc1840

61 And yet more roles for small RAs

62 RA σίγηση, ένας μηχανισμός ρύθμισης και άμυνας 1. History and definitions 2. The role of small RAs in gene regulation 3. Mechanisms of RA interference 4. Biological role of RA silencing 5. Applications of RA silencing 6. miras 7. RA-mediated epigenetic changes

63 Herve Vaucheret Genes Dev. 2006; 20:

64 Detection of of methylation in in DA Restriction enzyme cut no cut detection of HpaII CCGG CC M GG C M G MspII CCGG C M CGG C M G HaeIII GGCC GGC M C C M or bisulphite sequencing in combination with PCR

65 Discovery of of DA-dependent Pol PolIV IV in in plants

66 ature Reviews Genetics 4; (2003); doi: /nrg982 EXPLORIG PLAT GEOMES BY RA- IDUCED GEE SILECIG