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Structure and function of DICER

Structure and function of DICER. Presentation by Florian Ahrend. Overview. Introduction Function of RNAi Function of Dicer The RNase III – family Structure of Giardia -Dicer Giardia -Dicer vs Human-Dicer Flexibilty of Dicer The Argonaute – family RISC-loading Summary. Introduction.

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Structure and function of DICER

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  1. Structure and function ofDICER Presentation by Florian Ahrend

  2. Overview • Introduction • Function of RNAi • Function of Dicer • The RNase III – family • Structure of Giardia-Dicer • Giardia-Dicer vs Human-Dicer • Flexibilty of Dicer • The Argonaute – family • RISC-loading • Summary

  3. Introduction • What are Dicer and where do they occur? • multidomain proteins • about 200kDa • evolutionary highly conserved • occurs in a wide range of eukaryotes, including protozoa, invertebrates and plants (in prokaryotes -> RNase III) • involved in RNAi pathway (for gene-silencing)

  4. -central role of Dicer in RNAi pathway -cleaves 2nt-3´-overhang-dsRNA; endogenous (pre-miRNA) and exogenous dsRNA into miRNA and siRNA -products interact with RISC for sequence-specific gene-silencing

  5. Function of RNAi • for example: • chromatin remodeling • genome rearrangement • developmental timing • brain morphogenesis • stem cell maintenance • very important control mechanism for organism!

  6. Function of Dicer -Dicer initiates RNAi by processing dsRNA substrates -it is a molecular ruler, that measures and cleaves dsRNA into small fragments of discrete size (about 21-27 nucleotides in length) by hydrolysis Endoribonuklease activity -Dicer (of higher eukaryotes) aids to load produced short dsRNA into RISC (RNA-induced-silencing-complex)

  7. The RNase III - family RNase = Ribonuklease Dicer = specialized RNase Class 1: -simplest and smallest RNase III -consists of: single RNase III-domain + dsRBD (= dsRNA-Binding-Domain) -found in bacteria, bacteriophage and some fungi -forms homodimers

  8. The RNase III - family • Class 2: • termed “Drosha” • -consists of: tandem RNase III-domain + • dsRBD + • Polyproline-domain • -functions as monomer, while forming • an “internal dimer-structer”

  9. The RNase III - family • Class 3: • termed “Dicer” • consist of: tandem RNase III-domain + • dsRBD + • PAZ-domain (= Piwi-Argonaute-Zwille) + • DExD Helicase + • DUF283 (= domain of unknown function)

  10. Structure of Giardia-Dicer -Dicer from Giardia intestinalis is a kind of “minimal dicer”, with only core-functions -misses Helicase and DUF -processes dsRNA from helical end in a fashion similar to human Dicer -looks like a hatchet

  11. Structure of Giardia-Dicer Comparing Giardia-Dicer and Aquifex-RNase III

  12. Structure of Giardia-Dicer • PAZ-domain: • dsRNA-binding-module • (prefers 2nt-3´-overhang dsRNA) • approx. 150 amino acids • oligonucleotid-binding fold (OB) • connector-helix: • -single α-helix • -sets length of product-RNA

  13. Structure of Giardia-Dicer platform: -creates a large positively charged region -guides the dsRNA bridging domain: -connection between RNase IIIa and RNase IIIb

  14. Giardia-Dicer vs Human-Dicer Human-Dicer contains some additional domains, like the DUF283 and DExD-Helicase DUF283: -about 100 amino acids long -lies between helicase and PAZ-domain -in higher eukaryotes: probably platform function DExD-Helicase: -possible functions include unwinding products, translocating Dicer, fascilitating handoff of products

  15. Flexibility of Dicer Three rigid regions of Dicer (RNase-region, platform-region and PAZ-domain) linked by two hinges allows evolutionary flexibility.

  16. The Argonaute-family -evolutionary highly conserved -about 100kDa -consist of: PIWI-domain and PAZ-domain

  17. RISC-loading RISC-loading-complex needed, which consist of Argonaute, Dicer and TRBP Problem: Which strand of the dsRNA acts as guide for gene-silencing?

  18. Summary

  19. Literature -Macrae, I.J., et al., Structural basis for double-stranded RNA processing by Dicer. Science, 2006. 311(5758): p. 195-8. -MacRae, I.J. and J.A. Doudna, Ribonuclease revisited: structural insights into ribonuclease III family enzymes. Curr Opin Struct Biol, 2007. 17(1): p. 138-45. -Macrae, I.J., et al., Structure of Dicer and mechanistic implications for RNAi. Cold Spring Harb Symp Quant Biol, 2006. 71: p. 73-80. -Zhang, H., et al., Single processing center models for human Dicer and bacterial RNase III. Cell, 2004. 118(1): p. 57-68. -Skript zur Vorlesung “Genregulation”, Professor Nellen, Uni Kassel -http://www.rna.whu.edu.cn/Nucleic%20Acids%20structure%20and%20 biology/2005/6.ppt#24 -http://microrna.sanger.ac.uk/sequences -http://128.8.90.214/classroom/mocb639/J3JiangRNAi1.pdf -http://www.nature.com/ng/journal/v35/n3/full/ng1253.html

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