Messenger RNA Processing

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CHAPTER 15

• Messenger RNA Processing ?
• Capping and Polyadenylation
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  presented by luan rong

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Part 1 capping
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Reovirus cap structure
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• Digest the labled capping substance(xp) to yield
  x by phosphomonoesterase,then electrophoresis
  this digest.then,paper chromatography.

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Cap synthesis

• Sequence of events in capping?
• Verify the correct pathway
• Isolate each of the enzymes.
• ldentification of ppGpC as an intermediate in
  reovirus cap sythesis.

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(No Transcript)
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  caps and capping intermediates analysed
  by paper electrophoresis with marks .one
  radioactive intermediate coelectrophoresis with
  the ppGpC to GpC.then alkaline phosphatase
  convert ppGpC to GpC.
• Ion-exchang chromatography to identify ppGpC.

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Functions of caps

• Protection of the mrna from degradation(figure
  15.6)
• Enhancement of the mrna translatability.
• Tranport of mrna out of the nuleus.(figure 15.7)
• Proper splicing of the pre_mrna.

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• Labeled revirus rnas with capped, bloked,uncapped
  5end,subjected to glycerol gradient
  ultercentrifugation.
• Effect of incubation in xenopus oocytes and in
  wheat germ extrat decapping ,debloking

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Capping of u1 snrna is necessary for its
transport to the cytoplasm
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Part2 polyadenylation

• Size of poly(A) (generally 200bp)
• Functions of poly(A).(figure 15.10-12)
• Basic mechanism of polyadenylation.(figure
  15.14-26)

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Effect of polyadenylation on translatability and
stability of mrna
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Effect of polyadenylation on recruiment of mrna
to polysome
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Basic mechnism of polyadenylation

• Two hypothesis of the polyadenylation process.
• Polyadenylation signals.
• Cleavege and polyadenylation of a
  pre-mrna.(initiation-elongation)

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Two hypothesis of the polyadenylation of
adenovirus major late transcripts
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Importance of AAUAAA sequence to polyadenylation

• Mutant 1471 contains deplicated late
  polyadenylation sites 240bp apart.deletion either
  up/downstream site resulting in the loss of the
  coresponding aauaa sequence in the pre-mrna
  produced by mutant genes.

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Effect of rna POL2A and 2O on prepolyadenylation
mrna cleavage in vitro

• Incubate labeled adenovirus l3 pre-mrna with
  all the cleavage and polyadenyltion fators and
  polymerase 2A/O no protein(_) or purified HeLa
  cell SR protein.

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Effect of the RpB1CDT on prepolyadenylation mrna
cleavage in vitro
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A model for the precleavage complex
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Two fractions are needed for polyadenylation

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  Separation of PAP and
  specificity factor activity by DEAE-sepharose
  chromotography(100/600mM) from hela cell.
  Incubated them with ATP,then electrophoresed the
  labeled rna and autoradiographed the gel.

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Polyadenylation itsself consist of two phases

• Ues several different model RNA substrates
  performed in hela nulear extract.
• 1 58-nt substrate containing the 3-end of sv40
  late mrna.
• 2 the same rna with a 40-nt poly(A)
• 3 the same rna with A 40-nt 3-tag containing
  vector sequence.

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Demostration of two phases in polyadenylation
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Thus ,by the time 40-As have been added
polyadenylation is independent of AAUAAA signals

• Hypothesisthe shortest poly(A) that could
  override the effect of a mutation(AAUAAA)is 9 As
  which sugesst that after cleavage of the
  pre-mrna,polyadenylation depends on AAUAAA
  signals and CPSF until the poly(A) reaches about
  10 As in length.
• If CPSF recognizes the polyadenylation signals
  AAUAAA?

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CPSF binds to the AAUAAA motif
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Purification of poly(A) binding protein
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Effect of CPSF and PABIIon polyadenlylation of
model substrate
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Model for polyadenylation

• CPSF CstfF and CFI/II assemble on the pre-mrna
  guided by AAUAAA and GU/U motifs.
  cleavage occurs,stimulated by the CTP
  OF RNPIICstF,CFI/II leave the complex
  PAP enters .aided by CPSF
  initiates short poly(A) synesis.
• PABII enters the complex and allow the rapid
  extention of the oligo(A) to a full-length
  poly(A)

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