Structure and function of DICER

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Structure and function ofDICER

• Presentation by Florian Ahrend

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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

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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 -gt RNase III)
• involved in RNAi pathway
  (for gene-silencing)

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-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
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Function of RNAi

• for example
• chromatin remodeling
• genome rearrangement
• developmental timing
• brain morphogenesis
• stem cell maintenance
• very important control mechanism for organism!

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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)
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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
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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

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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)

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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
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Structure of Giardia-Dicer
Comparing Giardia-Dicer and Aquifex-RNase III
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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 a-helix
• -sets length of product-RNA

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Structure of Giardia-Dicer
platform -creates a large positively charged
region -guides the dsRNA bridging
domain -connection between RNase IIIa and RNase
IIIb
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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
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Flexibility of Dicer
Three rigid regions of Dicer (RNase-region,
platform-region and PAZ-domain) linked by two
hinges allows evolutionary flexibility.
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The Argonaute-family
-evolutionary highly conserved -about
100kDa -consist of PIWI-domain and PAZ-domain
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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?
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Summary
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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/Nucleic20Acids
20structure20and20 biology/2005/6.ppt24 -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