Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans

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Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis elegans(??)

• Jing Liu (??)

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Two features of RNAi in C.elegans that cant be
explained before 1998

• Sense and antisense RNA preparations are
  sufficient to cause interference separately.
• Interference effects can persist well into the
  next generation.
• This might reflect an underlying difference in
  RNA structure.

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Why predict dsRNA?

• Interference RNAstructure may be different from
  native RNA (eg.mRNA)
• The RNA polymerase used can produce some random
  or extopic transcripts.
• DNA transgene arrays also produce a fraction of
  aberrant(???) RNA products.

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How they test their prediction?

• They used several genes to test.
• Example unc-22 gene
• The gene produce a myofilament protein
• Decreases in unc-22 activity produce an
  increasingly severe twitching(??) phenotype.
• They injected sense, antisense, sense-antisense
  mixture into C.elegnats.

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• Unless a very high dose, the single strand RNA
  can produce any observable effect
• However, the mixture is at least two orders of
  magnitude(???) higer.
• The lowest dose of mixture that led to twitching
  phenotypes is about a few molecules per cell.

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• Electrophoretic analysis indicated that the
  injected material was predominantly
  double-stranded.
• Mix two kinds of single strand RNA at low salt
  concentration or inject them sequentially were
  sufficient for complete interference.
• But, a long interval(gt1h)between two RNA resulted
  a dramatic decrease in interfering activity.

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silencing was specific for an mRNA homologous to
the dsRNA

• Only related dsRNA can stimulate interference.
• They assess target specificty of dsRNA with
  unc-22 and three additional genes unc-54, fem-1,
  hlh-1, they all have well characterized
  phenotypes.
• Nearly all the phenotypic consequences of dsRNA
  injection were their expectation

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GFP were used to examine RNAi effect at a
cellular level

• Transgenic line PD4251 was used.
• Mosaic pattern was observed in the
  gfp-interference experiments, and this was
  nonrandom.
• The experiments indicated that some cells
  appeared to be resistant to interference.

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Control RNA (ds-unc22A)
ds-lacZL RNA
ds-gfpG RNA
a
d
g
e
h
b
c
f
i
i
a-c, progeny of animals injected with a control
RNA (ds-unc22A) d-f, progeny of animals injected
with ds-gfpG. d, a single active cell is seen in
the larva(??)e, the entire vulval musculature
expresses Active GFP in adult animal f two rare
GFP-positive cells in an adult g-i progeny of
animals injected with ds-lacZL RNA LacZ is
absent from almost all cells (see larva in g) h,
a typical adult, with nuclear GFP-lacZ Lacking
in almost all body-wall muscles but retained in
vulval muscles.
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some obersvations

• dsRNA segments corresponding to various intron
  and promoter sequences did not produce detectable
  interference(table1)
• The injection of dsRNA produces a pronounced
  decrease or elimination of the endogenous mRNA
  transcript(fig3)
• dsRNA-mediated interference showed a surprising
  ability to cross cellular boundaries(table2)

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Limitation

• A sequence shared between closely related genes
  may interfere with several members of the gene
  family
• A low level of expression will resist
  RNA-mediated interference for some or all genes.

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Thank you !

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Fig 3 effects of mex-3 RNA interference on levels
of the Endogenous mRNA. (A) Negative control
showing lack of staining(??) in the absence of
the hybridization probe. (B) Embryo from
uninjected parent showing normal pattern of
endogenous mex-3 RNA (C) Embryo from parent
injected with purified mex-3 antisense RNA. These
embryos (and the parent animals) retain mex-3
mRNA, although levels may be somewhat less than
wild type. (D) Late 4-cell stage embryo from a
parent injected with dsRNA corresponding to mex-3
no mex-3 RNA is detected.
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Table 2 Effect of site of injectionon
interference in injected animals and their progeny
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Gonad ??, Body cavity??