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Small RNA molecules and RNA interference in C.elegans

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Small RNA molecules and RNA interference in C.elegans Suvi Asikainen Kuopio University 27.8.2008 Introduction to C.elegans: Laboratory -course-messenger RNA (mRNA ... – PowerPoint PPT presentation

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Title: Small RNA molecules and RNA interference in C.elegans


1
Small RNA molecules and RNA interference in
C.elegans
Suvi Asikainen Kuopio University 27.8.2008
Introduction to C.elegans Laboratory -course
2
Ribonucleic acid (RNA)
RNA vs. DNA
RNA-world hypothesis
3
RNA species
  • -messenger RNA (mRNA)
  • -transfer RNA (tRNA)
  • -ribosomal RNA (rRNA)
  • -catalytic RNA (ribozymes)
  • -small interfering RNA (siRNA)
  • -ncRNAs (miRNA, tncRNA,piRNA...)

rRNA
www.contexo.info/DNA_Basics/Protein_synthesis.htm
4
Gene silencing by dsRNA
unc-22 dsRNA
progeny of injected worm
control
5
RNA interference (RNAi)
  • The Nobel Prize in Physiology or Medizine 2006
    for Craig Mello and Andrew Fire for their
    discovery of RNAi in C.elegans

nobelprize.org
6
RNAi mechanism in C.elegans basic scheme
  • less mRNA, less protein gene silencing

7
RNAi
  • conserved mechanism (at least 1 000 000 000
    (thousand billion) years old)
  • roles
  • primitive immune system
  • gene regulatory mechanism (via mRNA destruction)
  • chromatin remodelling (via histone modifications)
  • DNA elimination (in Tetrahymena thermophila)
  • tool
  • functional genomics
  • potential for pharmaceuticals

8
Different small RNA pathways in C.elegans
miRNA exogenous siRNA endogenous
siRNA
RRF-3, ERI-1
Duchaine et al., 2006. Cell
9
ERI complex for synthesis of endo-siRNAs in
C.elegans
  • proteins
  • Dicer (dsRNA specific endoribonuclease)
  • RRF-3 (RNA-dependent RNA polymerase)
  • ERI-1 (DEDH-type 5' exonuclease)
  • ERI-3 (novel protein)
  • ERI-5 (Tudor-domain protein)
  • mutant worm phenotypes enhanced exo-RNAi,
    sperm-defect in 25 ºC, high incidence of males
    due X-chromosome non-disjunction
  • the nature of precursor RNA molecule or gene/mRNA
    targets of the endo-siRNAs are not known

10
Working plan
miRNA exo-siRNA
endo-siRNA
loss of endo-siRNA molecules should lead to
accumulation of their mRNA targets
X
RRF-3, ERI-1
Duchaine et al., 2006. Cell
11
Microarray analysis of rrf-3 and eri-1 mutant
C.elegans
  • Affymetrix GeneChip -arrays
  • 3 array reps for each strain rrf-3, eri-1, N2

GeneSpring
12
Microarray analysis of rrf-3 and eri-1 mutant
C.elegans
Gene Ontology (molecular function) Count P-value
Protein kinase activity 17 22.08 3.43E-11
Phosphotransferase activity, alcohol group as acceptor 17 22.08 1.59E-10
Kinase activity 17 22.08 3.65E-10
Transferase activity, transferring phosphorus-containing groups 17 22.08 5.21E-09
Catalytic activity 32 41.56 5.32E-08
ATP binding 16 20.78 1.49E-07
Adenyl nucleotide binding 16 20.78 2.31E-07
Phosphoprotein phosphatase activity 9 11.69 2.64E-07
Nucleotide binding 17 22.08 1.53E-06
Purine nucleotide binding 16 20.78 1.57E-06
Phosphoric monoester hydrolase activity 9 11.69 1.69E-06
Phosphoric ester hydrolase activity 9 11.69 4.18E-06
Transferase activity 17 22.08 1.20E-05
Asikainen et al., 2007. FEBS Letters
13
Confirmation of results
  • quantitative RT-PCR
  • northern blot

Asikainen et al., 2007. FEBS Letters
14
Analysis of endo-siRNA sequences in silico
  • high throughput sequencing efforts by Lee et al.,
    2006 and Ruby et al 2006 have produced gt3000
    sequences of candidate endo-siRNAs freely
    available
  • functional categorization of mRNAs using GO terms
    (DAVID bioinformatics)
  • the length of siRNA correlates with a
    corresponding mRNA function!
  • 18- to 22-mer siRNAs were associated with
    embryonic development
  • 23-mers were associated with post-embryonic
    development
  • 24- to 26-mer siRNA sequences with phosphorus
    metabolism

15
Conclusions
  • over-expressed phosphorus metabolism and sperm
    associated transcripts in rrf-3 and eri-1 mutants
    are candidate targets for endo-siRNA pathway
  • 24- to 26-mer endo-siRNA sequences are candidate
    products of RRF-3
  • rrf-3 and eri-1 phenotypes might be result from
    misregulation of genes associated with phosphorus
    metabolism and sperm function

16
Acknowledgements!
  • Vuokko Aarnio
  • Kaja Reisner
  • Liisa Heikkinen
  • Markus Storvik
  • Jussi Paananen
  • Petri Pehkonen
  • Mitja Kurki
  • Tanel Jantsen
  • Richard Nass

Garry Wong Merja Lakso
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