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Seminar Molecular Maschines and RNA Biology

Seminar Molecular Maschines and RNA Biology. RITS Daniela Winkler 05. August 2007. Contents. (1) Definition of RITS (2) Overview RNAi (3) Components of RITS Role of Dicer (4) Role of Heterochromatin (5) Interaction of the components of RITS and of RITS and RDRC

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Seminar Molecular Maschines and RNA Biology

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  1. Seminar Molecular Maschines and RNA Biology RITS Daniela Winkler 05. August 2007

  2. Contents (1) Definition of RITS (2) Overview RNAi (3) Components of RITS • Role of Dicer (4) Role of Heterochromatin (5) Interaction • of the components of RITS and • of RITS and RDRC (6) RNAi-directed assembly of heterochromatin in fission yeast (7) RNAi-directed modification of heterochromatin

  3. Contents (8) siRNA-mediated recognition of specific chromosome regions (9) dsRNA synthesis (10) Chromosome-associated amplification of dsRNA (11) Recruitment of the histone H3-K9 methyltransferase Clr4 to chromatin (12) Conservation of RNAi components (13) Summary (14) References

  4. RITS • RNA-induced initiation of transcriptional gene silencing • a form of RNA interference by which short RNA molecules (miRNA or siRNA) trigger the downregulation of transcription • RITS has been studied extensively in the fission yeast Schizosaccharomyces pombe

  5. RNA interference • RNAi was initially defined by Fire et al. • process that is triggered by double strand RNA (dsRNA) and silences expression of genes in Caenorhabditis elegans • RNAi is conserved from Fungi to mammals • related to other RNA silencing processes [like quelling, co-suppression, and post-transcriptional gene silencing (PTGS)] • RNAi pathway plays important role in protecting cells against viruses and repetitive DNA elements • wide variety of cellular processes (regulation of development, growth, differentiation, apoptosis, and cancer) require the RNAi pathway

  6. RNA interference • Processing of double stranded RNA to small ~22 nucleotide interfering RNA (siRNA) by Dicer (ribonuclease III) • Dicer also processes cellular hairpin RNA, transcribed from the genome by RNA polymerase II, into micro RNAs (miRNAs) which are similar in size to siRNAs • Both types of small RNAs then act as specific factors that guide effector complexes to complementary sequences • RISC (RNA-induced silencing complex) uses small RNAs to target corresponding mRNAs for inactivation (through degradation or translational repression)

  7. RNA interference

  8. RNAi mostly functions at the posttranscriptional level to silence target mRNAs • In several cases at the DNA and chromatin level • Examples: • RNAi mediated DNA and histone methylation • RNAi is involved in massive DNA elimination that occurs in the somatic macronucleus of the protozoa, Tetrahymena

  9. RITS • downregulation of transcription is usually accomplished by modification of histones (by methylation or by the induction of heterochroamtin formation) • Protein complex that binds the miRNA and interacts with histones and DNA is known as the RITS complex • The RITS complex contains: • piwi-domain containing RNase-H-like argonaute • chromodomain protein Chp1 • Tas3 (required histone H3-K9 methylation) • additional: small RNAs

  10. Small RNAs • require Dicer for production • required for localization of RITS to heterochromatic domains (target recognition) • RITS-associated siRNAs are complementary to centromeric DNA repeats, where heterochromatin assembly is initiated

  11. Dicer-dependent association of RITS with siRNAs

  12. RITS • The RITS complex contains: • piwi-domain containing RNase-H-like argonaute • chromodomain protein Chp1 • Tas3 (protein of unknown function) • additional: small RNAs • Loss of genes result in abnormal heterochromatin organization and formation of the chromosome centromeres

  13. Heterochromatin • is silenced by conserved epigenetic modifications of histones and DNA • Epigenetic silencing and the packaging of repeats into heterochromatin is believed to • prevent illegitimate recombination (can lead to chromosomal rearrangements) • protect the chromosomes from active transposons (can cause mutations when integrated into genes)

  14. Heterochromatin • was thought to be inert • was discoverd to silence genes • its importance in regulating gene expression was controversial • is now known to give rise to small RNAs • Small RNAs direct the modification of proteins and DNA in heterochromatic repeats and transposable elements • has emerged as a key factor in epigenetic regulation of gene expression, chromosome stability and evolution

  15. RNAi-directed assembly of heterochromatin in fission yeast • The assembly of heterochromatin involves coordination of chromatin modifications and requires the RITS complex • Biochemical studies identified that RITS complex targets specific chromosome regions for inactivation • RITS regulates dsRNA and siRNA synthesis by recruiting a RNA-directed RNA polymerase complex (RDRC) • RNAi plays crucial role in heterochromatin assembly in S. pombe and other eukaryotes • Heterochromatic silencing in S. pombe occurs at the centromeres, telomeres and the mating-type loci

  16. Protein-protein interactions involving RITS and RDRC RDRC RITS Chp1 H3-K9 methyl Ago1 Tas3 Rdp1 Hrr1 Cid12 Spliceosome

  17. RNAi-directed assembly of heterochromatin in fission yeast • Purification of RNAi complexes RITS and RDRC provided insights into mechanism of RNAi-mediated heterochromatin assembly • Model: siRNAs act as factors that initiate epigenetic chromatin modifications and dsRNA synthesis at specific chromosome regions • Formation of heterochromatin involves different chromatin changes (including histone deacetylation, histone H3-lysine 9 methylation, and the recruitment of histone binding proteins such as Swi6/HP1) • Deacetylation of histone H3 amino termini by Clr3 and Clr6 (histon-deacetylases) is followed by the methyltransferase Clr4 to create a binding site for the Swi6 and Chp1

  18. RNAi-directed modification of heterochromatin • Methylation, acetylation, phosphorylation and ubiquitination of the core histones H2A, H2B, H3 and H4 are implicated in gene regulation • DNA methylation is nearly absent in yeast, flies and nematodes • DNA and histone modifications have a common role in gene silencing: they may even have a common origin • Silencing depends on the histonedeacetylases Clr6, the histone H3K9 methyltransferaseClr4, and on the HP1 homologue Swi6

  19. siRNA-mediated recognition of specific chromosome regions a) RITS-associated siRNAs directly base pair with DNA (require unwinding to allow base pairing) b) RITS is recruited indirectly to chromatin by base pairing interactions between siRNAs and nascent RNA transcripts at the target locus Two models how RITS targets specific chromosome regions Nucleosome methylatet lysine 9 of H3

  20. siRNA-mediated recognition of specific chromosome regions • Recent results favor b) model • Prediction is that RITS should associate with transcripts at the target locus in a siRNA-dependent way • RNA immunoprecipitation experiments show that RITS associates with non-coding centromeric RNAs but not with actin mRNA (euchromatin gene) • Association of RITS with non-coding centromeric RNA is Dicer- and therefore siRNA-dependent • RITS localization to chromatin requires methylation of H3-K9 by Clr4 (thought to stabilize the association of RITS with chromatin by creating a binding site for the chromodomain of Chp1)

  21. dsRNA synthesis • generation of siRNA requires a source of dsRNA • centromeric dsRNA could originate from two different sources: • (i) transcription of sense and anti-sense RNA from both DNA strands • (ii) the synthesis of a complementary RNA from an RNA template by an RNA-directed RNA polymerase (RDR) • In plants, an RDR is required for histone and DNA modifications, amplification of siRNAs by autoregulatory loops • Fission yeast has one RDR, Rdp1, which associates with centromeric heterochromatin (in rdp1- cells dsRNA is produced by transcription of both centromeric DNA strands, no siRNAs detected in RITS)

  22. Chromosome-associated amplification of dsRNA • Rdp1 plays a primary role in producing siRNAs • transcription of both centromeric DNA strands results in production of a low amount of dsRNA and siRNA • initial siRNA then recruit Rdp1 to homologous RNA transcripts, resulting in the amplification of dsRNA and the production of larger amounts of siRNA • yeast Rdp1 is associated with two other proteins, Hrr1, a putative RNA helicase, and Cid12, a putative polyA polymerase, in a complex termed the RDRC

  23. Chromosome-associated amplification of dsRNA • RDRC has an RNA-directed RNA-polymerase activity, which is required for siRNA generation and silencing in vivo • RDRC and RITS physically interact (Dicer and Clr4 dependent) • In dcr1- cells RITS is not loaded with siRNAs (no localization to centromeric repeats, centromeric heterochromatin does not form) • Clr4 regulates dsRNA and siRNA production by controlling the interaction of RDRC with RITS • Clr4 is a homolog of the human and Drosophila Suv39h methyltransferases, required for histone H3-K9 methylation

  24. Recruitment of the histone H3-K9 methyltransferase Clr4 to chromatin • In clr4-cells purified RITS contains little or no siRNAs • Clr4 perhaps regulates siRNA generation by promoting the association of RITS and RDRC with target chromatin regions • By methylating H3-K9, Clr4 allows RITS to bind to chromatin through interactions with both the nascent transcript via siRNAs, and methylated nucleosomes via the chromodomain of Chp1

  25. Recruitment of the histone H3-K9 methyltransferase Clr4 to chromatin • In clr4- cells, RITS and RDRC no longer interact and Rdp1 fails to associate with centromeric DNA or RNA. • Mutations in the chromodomain of Chp1 disrupt its interaction with methylated lysine K9 of histone H3 also dramatically reduce the amount of siRNA in RITS • Suggestion: dsRNA amplification occurs on the chromosome and is regulated by Clr4-dependent H3-K9 methylation

  26. Conservation of RNAi components • C. elegans has 24 Argonaute genes • Although RDR enzymes are conserved in plants and C. elegans, they appear to be absent in Drosophila and mammals • Perhaps RNA polymerase II can use RNA as a template and produce dsRNA

  27. Summary • heterochromatic silencing depends on the processing of repeat RNA transcripts into short interfering RNAs (siRNAs), which then direct chromatin modification • RITS provides a direct physical link between RNAi and heterochromatin assembly • heterochromatic silencing is important in evolution and development

  28. References • Buhler, M., A. Verdel, and D. Moazed, Tethering RITS to a nascent transcript initiates RNAi- and heterochromatin-dependent gene silencing. Cell, 2006. 125(5): p. 873-86 • Lippman, Z. & Martienssen, R. The role of RNA interference in heterochromatic silencing. Nature 431 (2004) 364-370 • Motamedi, M.R., et al., Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell, 2004. 119(6): p. 789-802 • Verdel, A., et al., RNAi-mediated targeting of heterochromatin by the RITS complex. Science, 2004. 303(5658): p. 672-6 • Verdel, A., Moazed, D.: RNAi-directed assembly of heterochromatin in fission yeast. FEBS Letters 579 (2005) 5872–5878 • http://www.wikipedia.org

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  30. A speculative model for recruitment of dsRNA amplification machinery (RDRC) and the Rik1–Clr4–Cul4 complex to the nascent transcript by the RITS complex • Possibly Rik1 (a heterochromatin protein) directly links the RNAi machinery to histone H3-K9 methylation through the recognition of a specific nucleic acid substrate generated during siRNA targeting of specific chromosome domains. • it remains unclear how RNAi recruits Clr4 to chromatin

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