Systematic RNAimediated mRNA knockdown of a large

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Systematic RNAi-mediated mRNA knockdown of a
large subset of genes on T. brucei Chromosome 1.
Similar approach taken in yeast, nematode, insect
and human
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Challenge Annotation of Genome Sequencing Data
Trypanosoma brucei, Trypanosoma cruzi and
Leismania major Genomes recently sequenced (2005).

• 76 of T. brucei genes are shared between
• all three sequenced kinetoplastids.

• Over half of predicted trypanosome ORFs have no
• counterpart in higher eukaryotes.

• 73 of trypanosome ORFs have unknown,
• poorly predictable or unpredictable functions.

• Only T. brucei has a robust RNAi pathway.
• Well characterized Tet inducible RNAi expression.

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Trypanosome Lifecycle
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RNAi Overview

• Method for ablation of specific mRNA
• Depends on double-stranded RNA
• Exogenous (transfected dsRNA)
• Endogenous (DNA expression vector)

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Strategy

• Select genes for knock-down by RNAi
• Produce gene-specific Tet inducible RNAi
  constructs
• Transfect constructs into bloodstream form T.
  brucei
• Score phenotype following RNAi induction
• Growth and viability
• Cell cycle progression
• Endocytic and protein processing systems
• Morphology and Motility
• Post results on publicly accessible database
• Rigorous quality control

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Organization of Chromosome 1
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Criteria Used to Select Genes for RNAi Knockdown
369 predicted ORFs on Chromosome 1 (8131 ORFs on
11 chromosomes)

• Chose 197 ORFs for RNAi ablation
• Excluded subtelomeric regions (300kb)
• Gene clusters specific for Chromosome 1
• Pan-specific RNAi targeting all members
• Excluded multi-gene family members present
• on other chromosomes
• Excluded those that failed RNAit algorithm
  criteria
• 2 gave no PCR product

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RNAi Expression Constructs
Transfected into BF Lister 427 expressing TetR
and T7 Pol

• Concerns
• Leaky expression
• Expression too low (not knocked-down enough)
• Variable expression at different integration sites

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Growth and Viability

• Identical transfected cultures were seeded at
  1x106 cells/ml
• one culture induced with 1µg/ml tetracycline
• Cell number counted daily and cultures reseeded
  at
• 1x106 cells/ml for up to 8 days.
• Defect scored if density lt 75(mild) or
  50(severe) less than
• uniduced culture.

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Cell Cycle Progression
Microscopic assessment of nucleus and kinetoplast
number following DAPI staining. WT cells 85
1K1N, 10 2K1N, 5 2K2N Defect lt4 or
gt17 2K1N gt12 2N gt4.5 0N 0K Ngt2 Kgt2
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Endocytic and Protein Processing Systems

• Concanavalin A (ConA) reacts with VSG
  flagellar pocket,
• endosomes and lysosomes.
• Tomato Lectin glycoproteins
• MitoTracker mitochondria
• BODIPY-ceramide Golgi

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Endocytic and Protein Processing Systems
TFN1.195

• Defective ConA uptake
• Phase-light vacuole

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Data Summary

• 33 showed significant phenotype.
• 23 growth defect 16 cell cycle progression 6
  both.
• Only one showed additional defects in endocytic
  pathway.
• Visible defects in endocytic pathway and
  morphology almost
• invariably associated with growth/cell cycle
  defects.
• No evidence of functional clustering strand bias
  or positional
• effects that correlate with knock-down phenotype.

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Hows the current annotation?
47/120
Genes annotated as function inferred from
homology and conserved hypothetical are
generally accurate.
Genes annotated as hypothetical and
hypothetical, unlikely are less likely to
represent functional ORFs.
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Are genes widely conserved between species
more likely to be essential for viability and
growth than genes conserved within a closely
related group?

• Core functions versus life cycle-specific
  functions.

• Appears to be the case in S. cerevisiae and S.
  pombe.

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Are genes broadly conserved between species more
likely to be essential for viability and growth?
No statistically significant difference in T.
brucei.
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Conclusions

• 12 of knockdowns lethal further 11 growth
  inhibited.
• 1,500 ORFs for robust growth in vitro.
• No higher order genome organization associated
  with
• knockdown phenotype.
• No increased frequency of knockdown phenotype
• associated with widely conserved genes.
• Other genes may be essential under different
  conditions/
• lifecycle stages.
• Some may be missed due to technical issues.
• Growth / cell cycle phenotypes are most robust
  and telling.
• May be most useful for rapid screens.
• New methods must be developed to determine
  functions of
• those genes with inconclusive knockdown
  phenotypes.

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Future Directions

• Determine function/ biochemical activity of
  essential
• proteins with unknown function.
• High throughput localization.
• Live-cell reporter strains (FP tagged organelles)
• Continue analysis with ORFs on 10 remaining
  chromosomes.
• High throughput analysis using RNAi barcodes.
• Couples RNAi libraries with microarray analysis.