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Seminar Molecular Machines & RNA Biology

Seminar Molecular Machines & RNA Biology. RNA Polymerase IV. Jennifer Hermann. Content. Introduction: RNA Polymerases

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Seminar Molecular Machines & RNA Biology

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  1. Seminar Molecular Machines & RNA Biology RNA Polymerase IV Jennifer Hermann

  2. Content • Introduction: RNA Polymerases • Plant Nuclear RNA Polymerase IV Mediates siRNA and DNA Methylation-Depentdent Heterochromatin Formation (Yasuyuki Onodera,Jeremy R. Haag, Thomas Ream, Pedro Costa Nunes, Olga Pontes and Craig S. Pikaard1) Cell, Vol. 120, 613-622, March 11, 2005

  3. Content • RNA Polymerase IV Directs Silencing of Endogenous DNA (A. J. Herr, M. B. Jensen, T. Dalmay, D. C. Baulcombe) Science, 2005. 308(5718): p. 118-20 • Role of RNA polymerase IV in plant small RNA metabolism(Zhang, X., et al.) Proc Natl Acad Sci U S A, 2007. 104(11): p. 4536-41 • conclusions • table of figures • references

  4. Introduction • RNA polymerase • definition: RNA polymerase (RNAP or RNApol) is an enzyme that makes a RNA copy of a DNA or RNA template (wikipedia.com; 07/2007) • 1960: RNAP was discovered independently by Sam Weiss and Jerard Hurwitz • 2006: Nobel Prize in Chemistry was awarded to Roger Kornberg for creating detailed molecular images of RNA polymerase during various stages of the transcription process Fig. 1

  5. Introduction • constructing RNA chains from DNA genes → transcription • in chemical terms: RNAP is a nucletidyl transferase • Products of RNAP include: • mRNA • Non-coding RNA or “RNA-genes” • tRNA • rRNA • microRNA • ribozymes Fig. 3 Fig. 2 Fig. 4 Fig. 5

  6. Introduction – RNA polymerase • RNAP accomplishes de novo synthesis • prefers to start transcripts with ATP • includes helicase activity Fig. 6: RNAP from T. aquaticus

  7. Introduction – RNA polymerase action • switch from a closed to an open complex • Ribonucleotides are base-paired to the template DNA strand • supercoiling plays an important patr in polymerase activity • elongation: • involves the further addition of ribonucleotides • change of the open complex to the transcriptiomal complex • very similar mechanism to DNA polymerisation Fig. 7: an electron-micrograph of DANN strands decorated with hundreds of RNAP molecules

  8. Introduction – RNA polymerase in bacteria • relatively large molecule • consists of 5 subunits • α1,2 • α-CTD • α-NTP • β: catalyzes the synthesis of RNA • β`: binds to DNA • ω: protective / chaperone function • another subunit σ is required for binding to promotor specific regions Fig. 8

  9. IntroductionRNA polymerase in archaea; viruses • archaea: • single RNAP • related to the main three eukaryotic polymerases → resembles the anchestor of the specialized eukaryotic polymerase • viruses: • most widely studied viral RNAP is found in bacteriophage T7 • Related to that found in mitochondria and chloroplasts Fig. 9 Fig. 10

  10. Introduction RNA polymerase eukaryotes • 3 nuclear DNA-dependent RNA polymerases transcribe genomic DNA into RNA • RNA polymerase I • transcribes rRNA genes • RNA polymerase II • transcribes the vast majority of genes • RNA polymerase III • Transcribes genes encoding short structural RNAs • composed of 12-17 proteins Fig. 11: Structure of an RNA polymerase II transcribing complex

  11. Introduction RNA polymerase eukaryotes • Pol I, II and III • yeast: RPA, RPB, RPC • Arabidopsis: N[nuclear]RPA, NRPB, NRPC • largest subunit: • homologous to eubacterial β` • encoded by different genes: • (N)RPA1, (N)RPB1, (N)RPC1 • second largest subunit: • β homologs • encoded by: • (N)RPA2, (N)RPB2, (N)RPC2 → together: catalytic center

  12. Plant Nuclear RNA Polymerase IV mediates siRNA and DNA Methylation-Dependent Heterochromatin Formation • analysis of the Arabidopsis thaliana genome sequence: • evidence for a fourth class of RNA polymerase • distinct from eubacterial-type RNAPs of chloroplasts • mitochondrial polymerase • RNA-dependent RNA polymerases (RdRP) • evidence: • RNA Pol IV is located within the nucleus • plays a role in heterochromatin formation • propose that Pol IV is required for the production of siRNAs

  13. Plant Nuclear RNA Polymerase IV mediates siRNA and DNA Methylation-Dependent Heterochromatin Formation • Unrooted phylogenetic tree of RNAP largest an second-largest subuntit • Arabidopsis thaliana (At) • rice (Os) • yeast (Sc; Sp) • C. elegans (Ce) • Drosophila (Dm) • human (Hs) Fig. 12

  14. Heterochromatin Association Is Impaired in nrpd2 Mutants • nrpd2 mutants: • increased number and decreased size of DAPI-positve heterochromatic foci • H3dimethylK9 signals are dispersed; colocalize with small DAPI-positive foci • Chromocenters involving NORs are relatively resistant to dispersal • nrpd double mutant siblings • 5S genes are decondensed • less colocalization Fig. 13 Fig. 14

  15. Pol IV participates in the siRNA-Chromatin Modification Pathway • Heterochromatin disruption; 5S gene dispersal in Pol IV mutants • loss of cytosin methylation • methylation sensitive restriction endonucleases • HpaII (methylation at the inner C: no activity) • MspI (methylation at the outer C: no activity) → cut CCGG motifs • HaeIII (methylation at the inner C: no activity → cut GGCC motifs Fig. 15

  16. Pol IV participates in the siRNA-Chromatin Modification Pathway • DRM2:responsible for de novo methylation • DDM1:involved in maintenance of methylation • MET1:responsible for maintenance of CG methylation • DRM1:no known function • CMT3:responsible for maintenance of CNG methylation Fig. 15 → Pol IV affects 5S gene methylation in all sequence contexts

  17. Pol IV participates in the siRNA-Chromatin Modification Pathway • the highly methylated 180 bp centromere repeats are unaffected by nrpd1 and nrpd2 mutations → Pol IV does not affect global cytosine methylation levels Fig. 16

  18. Pol IV participates in the siRNA-Chromatin Modification Pathway • Methylation of AtSN1 • HaeIII digestion followed by PCR • Wild-type Col-0, Ler, Ws • AtSN1 heavily methylated → resistent to HaeIII cleavage • Met1, cmt3: unaffected • Drm1drm2: reduced Fig. 17 → HaeIII methylation is also disrupted in mutants of heterochromatic siRNA pathway → AtSN1 methylation reduced in both nrpd1 and nrpd2 mutants

  19. RNA Polymerase IV Directs Silencing of Endogenous DNA • identified an Arabidopsis nrpd1a-1 mutant • exhibit partial loss of transgene silencing pathway • defective for • siRNA production • methylation of the SINE retroelement AtSN1 • used an Arabidopsis line • a GFP transgene was silenced by a potato virus X (PVX)-GFP transgene

  20. RNA Polymerase IV Directs Silencing of Endogenous DNA • delayed onset of silencing in growing points of the plant • to complement the GFP silencing phenotype of nrpd1a-1 failed Fig. 18 Fig. 19

  21. RNA Polymerase IV Directs Silencing of Endogenous DNA • the patterns of transgene mRNA and siRNA accumulation corresponded to the amount of GFP-fluorescence • northern analysis • nrpd1a-1 • increased accumulation of • GFP-mRNA (4.5 fold) • PVX-GFP RNA Fig. 20

  22. RNA Polymerase IV Directs Silencing of Endogenous DNA • RNA-directed DNA methylation (RdDM) is associated with silencing in plants • GFP DNA methylation, GFP siRNAs • rdr6 (both absent) • sgs3 (both absent) • nrpd1a-1 (reduced, slight changes) Fig. 21

  23. RNA Polymerase IV Directs Silencing of Endogenous DNA Fig. 22 • characterized another mutant with a delayed onset of silencing • inversion on chromosome 4 that disrupts RDR2 • lower amounts of endogenous 24-nt siRNAs • miR167 amounts are unaffected • likely Pol IV, RDR2, DCL3 act together in a silencing pathway Fig. 23

  24. Role of RNA polymerase IV in plant small RNA metabolism • RNA Pol IV appears to be specialized in the production of siRNAs • model: • dsRNA are generated by RNAP IV & RDR2 • processed by DCL enzymes into 21- 24 nt siRNAs • associated with different AGOs • not yet clear what fraction of genomic siRNA production is RNAP IV dependent

  25. Role of RNA polymerase IV in plant small RNA metabolism- 454 – technology (454 Life SciencesTM) Fig. 24 • DNA library preparation • emPCR • sequencing

  26. Role of RNA polymerase IV in plant small RNA metabolism • RNAP IV is required for the production of 90% of all siRNAs • strong similarity among the profiles of RNAP IV & RDR2 • only 9,7% of individual siRNA reads • 54,6% of 21mers • 84% of 22 mers • 98,9 % of 24 mers → were lost in nrpd1a/1b Fig. 25

  27. Role of RNA polymerase IV in plant small RNA metabolism • as a consequence • the most abundant size: 21 mers • 24mers were sparse • the ratio became 21 : 22 : 24 1 : 0.43 : 0.18 • wt: 1 : 1.25 : 7.59 Fig. 26

  28. conclusions • RNAP IV is required for the production of >90% of all siRNAs • likely Pol IV, RDR2, DCL3 act together in a silencing pathway • Pol IV helps produce siRNAs that target de novo cytosine methylation events • required for: • facultative heterochromatin formation • high-order heterochromatin association

  29. Thanks for your attention! Science consists in grouping facts so that general laws or conclusions may be drawn from them Charles Robert Darwin

  30. table of figures • cover: http://www.biology.wustl.edu/faculty/FacultyPage.php?IDProf=26 (An Arabidopsis nucleus showing the relative locations of RNA polymerase IV (green) and RNA polymerase II (red)) • Fig.1; 11: Advanced information on the Nobel Prize in Chemistry 2006; Molecular basis of eukaryotic transcription • Fig. 2: http://en.wikipedia.org/wiki/Image:Schema_ARNt_448_658.png • Fig. 3: http://en.wikipedia.org/wiki/Image:3d_tRNA.png • Fig. 4: http://www.steve.gb.com/science/genomes.html; human 5S rRNA • Fig. 5: Functional Hammerhead Ribozymes Naturally Encoded in the Genome of Arabidopsis thaliana (Rita Przybilski, Stefan Gräf, Aurelie Lescoute, Wolfgang Nellen, Eric Westhof, Gerhard Steger and Christian Hammann) • Fig. 6: http://upload.wikimedia.org/wikipedia/en/9/98/RNAP_TEC_small.jpg • Fig. 7: http://upload.wikimedia.org/wikipedia/en/a/aa/Transcription_label_fromcommons.jpg • Fig. 8: http://fig.cox.miami.edu/~cmallery/150/gene/sf13x5a.jpg • Fig. 9: http://upload.wikimedia.org/wikipedia/en/0/01/RNA_pol.jpg • Fig. 10: http://www.biologie.uni-hamburg.de/lehre/bza/virus/1rdr/drosette.jpg • Fig. 12: Fig. 1A in Plant Nuclear RNA Polymerase IV Mediates siRNA and DNA Methylation-Depentdent Heterochromatin Formation • Fig. 13: Fig. 1E in Plant Nuclear RNA Polymerase IV Mediates siRNA and DNA Methylation-Depentdent Heterochromatin Formation • Fig. 14: Fig. 2A-C in Plant Nuclear RNA Polymerase IV Mediates siRNA and DNA Methylation-Depentdent Heterochromatin Formation • Fig. 15: Fig. 4A in Plant Nuclear RNA Polymerase IV Mediates siRNA and DNA Methylation-Depentdent Heterochromatin Formation • Fig. 16: Fig. 4B • Fig. 17: Fig. 4C • Fig. 18: Fig. 1A RNA Polymerase IV Directs Silencing of Endogenous DNA • Fig. 19: Fig. S1F RNA Polymerase IV Directs Silencing of Endogenous DNA, supplementary data • Fig. 20: Fig. 1B in RNA Polymerase IV Directs Silencing of Endogenous DNA • Fig. 21: Fig. 1C • Fig. 22: Fig. S4B: RNA Polymerase IV Directs Silencing of Endogenous DNA, supplementary data • Fig. 23: Fig. 3A: RNA Polymerase IV Directs Silencing of Endogenous DNA • Fig. 24: http://www.454.com/enabling-technology/ • Fig. 25: Role of RNA polymerase IV in plant small RNA metabolism, table 2 • Fig. 26: Role of RNA polymerase IV in plant small RNA metabolism, Fig. 1 • DAPI: http://en.wikipedia.org/wiki/DAPI

  31. references • Advanced information on the Nobel Prize in Chemistry 2006;Molecular basis of eukaryotic transcription • Plant Nuclear RNA Polymerase IV Mediates siRNA and DNA Methylation-Depentdent Heterochromatin Formation (Yasuyuki Onodera,Jeremy R. Haag, Thomas Ream, Pedro Costa Nunes, Olga Pontes and Craig S. Pikaard1) Cell, Vol. 120, 613-622, March 11, 2005 • RNA Polymerase IV Directs Silencing of Endogenous DNA (A. J. Herr, M. B. Jensen, T. Dalmay, D. C. Baulcombe) Science, 2005. 308(5718): p. 118-20 • Role of RNA polymerase IV in plant small RNA metabolism(Zhang, X., et al.) Proc Natl Acad Sci U S A, 2007. 104(11): p. 4536-41 • www.wikipedia.com • www.454.com/enabling-technology/

  32. DAPI: 4',6-diamidino-2-phenylindole • fluorescent stain that binds strongly to DNA • used extensively in fluorescence microscopy • DAPI will pass through an intact cell membrane • it may be used to stain both live and fixed cells • fluorescence microscopy • DAPI is excited with ultraviolet light • absorption maximum is at 358 nm; emission maximum is at 461 nm. • DAPI also bind to RNA (not as strongly fluorescent) • emission shifts to around 500 nm when bound to RNA • DAPI's blue emission is convenient for multiple fluorescent stains in a single sample. • fluorescence overlap between DAPI • green-fluorescent molecules like fluorescein • green fluorescent protein (GFP) • red-fluorescent stains (like Texas Red) • labelling cell nuclei, detection of mycoplasma or virus DNA in cell cultures • DAPI readily enters live cells and binds tightly to DNA, it is toxic and mutagenic

  33. FISH (Fluorescent in situ hybridization) • cytogenetic technique • can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes • uses fluorescent probes which bind only to those parts of the chromosome with which they show a high degree of sequence similarity • Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosome • FISH is often used for finding specific features in DNA • These features can be used in genetic counseling, medicine, and species identification

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