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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

Structure and function ofDICER

Presentation by Florian Ahrend

overview
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
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)
slide4

-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

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

the rnase iii family
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

the rnase iii family8
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”
the rnase iii family9
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)
structure of giardia dicer
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

structure of giardia dicer11
Structure of Giardia-Dicer

Comparing Giardia-Dicer and Aquifex-RNase III

structure of giardia dicer12
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
structure of giardia dicer13
Structure of Giardia-Dicer

platform:

-creates a large positively charged region

-guides the dsRNA

bridging domain:

-connection between RNase IIIa and RNase IIIb

giardia dicer vs human dicer
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

flexibility of dicer
Flexibility of Dicer

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

the argonaute family
The Argonaute-family

-evolutionary highly conserved

-about 100kDa

-consist of: PIWI-domain and PAZ-domain

risc loading
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?

literature
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