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Seminar Molecular Machines & RNA Biology. RNA Polymerase IV. Jennifer Hermann. Content. Introduction: RNA Polymerases
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Seminar Molecular Machines & RNA Biology RNA Polymerase IV Jennifer Hermann
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
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
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
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
Introduction – RNA polymerase • RNAP accomplishes de novo synthesis • prefers to start transcripts with ATP • includes helicase activity Fig. 6: RNAP from T. aquaticus
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Role of RNA polymerase IV in plant small RNA metabolism- 454 – technology (454 Life SciencesTM) Fig. 24 • DNA library preparation • emPCR • sequencing
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
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
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
Thanks for your attention! Science consists in grouping facts so that general laws or conclusions may be drawn from them Charles Robert Darwin
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
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/
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
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