MicroRNA Inhibition of Translation Initiation in Vitro by Targeting the CapBinding Complex eIF4F

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MicroRNA Inhibition of Translation
Initiation in Vitro by Targeting the Cap-Binding
Complex eIF4F

• Liang kai wei
• Du tian peng

Oct.31
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• Background of miRNA
• Plant and animal genomes contain an
  aboundance of small genes that produce RNAs of 22
  nucleotides in length
• which were dubbed as microRNAs.These newly found
  endogenous RNAs may participate in a wide range
  of genetic regulatory pathways and play an
  important role in the development..

Fig.1
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MicroRNAs control gene expression in three ways

• 1lead to the degradation of the target mRNA
• 2 Inhibit translation
• 3 Proteolysis of the nascent polypeptide

Flash
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Limitation of microRNA inhibiting of translation
control

• In vivo studies ,the outcome of mRNA
  silencing has been examined hours or days after
  the initial mRNA target recognition.So the
  development of an vitro system is necess -ary to
  understand the biochemistry of miRNA function
  ,especially the early steps after the recruitment
  of RISC to the mRNA.

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To solve the problem

• Chose an in vitro translation system which can
  support efficient translation and exhibit many
  endogenous and viral translation control
  mechanism ,and the most important is the vitro
  system can faithfully recapitulate the property
  of miRNA.

Better chose the mouse Krebs-2 ascites
cell extract
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Concept

• Report gene renilla luciferase
• RL
• RL-6xB

used as control
contains six target sites for let-7 miRNA

• RL-6xBMut contains mutated site which let-7

can not recognize
Let-7miRNAs have many types,including let-7a
and let-7f miRNA
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Part1Experiments show the target mRNA
repression is sensitive to the relative
concentrations of the mRNA and miRNA

• EXP1. mRNAs were translated at concentrations
  varying from 3 pM to 3nM.At the lowest
  concentration (3pM),RL-6xB mRNA translation was
  only 25of RL mRNA.

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EXP2.A decrease in translation was not observed
at an mRNA concentration of 3nM with a limiting
concentration of let-7 miRNA
S1
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EXP3.The same degree of translation inhibition
was observed when Rluc activity was observed when
Rluc activity was normalized against firefly
luciferase(Fluc) activity(expressed from Fluc
mRNA used as an internal control)
?
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EXP4.RL and RL-6xB mRNAs were translated with
similar efficiency in a wheat gem extract when
let-7 is absent.
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Part2Experiments address the specificity of the
inhibition of RL-6xB mRNA translation by let-7

• .

• Exp1.Supplement the Krebs-2 ascites extract with
  an antisense 2-O-methyl(2-O-Me)
  oligoribonuclotide complementary to

let-7 miRNA.This resulted in an 2.5-fold increase
in translation of RL-6xB mRNA.

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Exp2.Translation of a reporter mRNA containing
mutations (RL-6xBMut) was almost as efficient (90
) as RLmRNA.This exp shows the importance of the
let-7 seed region (six target site) for
inhibition of mRNA expression for inhibition of
mRNA expression
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• Part1 and part2 indicate that the Krebs-2 ascites
  cell-free translation system faithfully
  recapitulate the properties of miRNAs establishde
  in vitro.

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Part3 Experiments indicate that mRNA degradation
is not responsible for the inhibition mediated by
let-7 seen as early times

• Exp1.Kinetics of let-7 mediated inhibition
  revealed a decrease(25)in translation of RL-6xB
  mRNA relative to RL mRNA,frist detected after 15
  min of incubationa stronger inhibition occurred
  at later times.

Fig7
Exp2
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Fig.2
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Fig.1