Messenger RNA Processing

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• CHAPTER 15
• Messenger RNA Processing ?
• Capping and Polyadenylation
• fan loukong (???)

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• This chapter includes 3 parts
• 1.Capping
• 2.Polyadenylation
• 3.Coordination of mRNA Processing Events

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Capping

• 1.Cap Structure
• 2.Cap Synthesis
• 3.Functions of Caps

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Cap Structure

• 1.The first caps to be characterized came from
  viral RNAs.Bernard Moss and his colleagues
  produced vaccinia virus mRNA labeled with 32p
  in vivtro and used DEAE-cellulose chromatography
  to isolated the Caps.
• 2.The researchers found they could label the Cap
  with ß,?-32pATP(but not with ?-32pATP).

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• So they put forward the Cap was at the
  5-terminus of the RNA. Because the ß-phosphate
  of a nucleoside triphosphate remains only in the
  first nucleotide in an RNA.
• They also think there must be some substance (x)
  to protect or block the ß-phosphate, because the
  alkaline phosphatase cant remove it.
• What is x?

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Block agent Xp X

• The researchers use phosphodiesterase to remove
  block agent to get Xp,and use phosphomonoesterase
  to get X.
• Then they subject this digest to paper
  electrophoresis,followed by paper chromatography.
  
• The figure 15.2 shows the X is m7G

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• Another product of phosphadiesterase cleavage of
  the cap was pAm(2-o-methyl-AMP),thus m7G is
  linked to pAm in the cap.
• But whats the nature of the linkage?
• The following three considerations tell us it is
  a triphosphate and the linkage is very likely to
  be 5to 5 1.the ?-phosphate,but not the
  ß-or?-phosphate of GTPwas remained in the cap.
• 2. the ß-and ? -phosphates of ATP are remained in
  the cap .
• 3. Both ATP and GTP have their phosphates in the
  5-position.

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Cap Synthesis

• The researchers studied capping of model
  substrates in vitro, they used cores from
  vaccinia virus and reovirus ,respectively,to
  provide the capping enzymes. in both viruses,
  they observed the some sequence of events, as
  illustrated in the figue15.4

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• To verify the pathway is correct, the researchers
  added 14CCTP and 32PGTP to reovirus cores to
  label the caps (pppGpC as a example) and capping
  intermediates ,then they analyzed the mixture by
  paper electrophoresis with markers listed at the
  top.

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• Summary
• Caps are made in steps
• first, an RNA triphosphatase removes the
  terminal phosphate from a pre-mRNAnext,a
  guanylyl transferase adds the capping GMP (from
  GTP). Next, two methyl transferase methylate the
  N7 of the capping guanosine and the 2-o-methyl
  group of the penultimate nucleotide

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Functions of Caps

• 1.Protection
• The cap is expected to protect the mRNA from
  attack by RNase that begin at 5-end of their
  substances and that cannot cleave triphosphate
  linkage.
• The figure 15.6 showed the cap can make the RNA
  more stability.

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• 2.Another important function of the cap is to
  provide translatability(???),we will see in
  chapter 17 that mRNA gains access to the ribosome
  for translation via a cap-binding protein.
• 3.The cap also appears to facilitate the
  transport of a mature RNA out of the nucleus.
• 4.People put forward that the cap is essential
• for proper splicing of a pre-mRNA

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Polyadenylation

• To get and purify Hela cell poly(A) from the rest
  of the mRNA molecule,researchers released it with
  two enzymesRNase A,which cuts after C and U,and
  RNase T1 ,which cuts after G.In other words
  they cut every nucleotide except the
  As,preserving only pure runs of As.
• Next they electrophoresed the poly(A)s from
  nuclei and from cytoplasm to determine their
  sizes,the figure15.8 shows the size of poly(A)
• .

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• It is apparent that the poly(A) goes on the
  3-end of the mRNA or hnRNA,because it can be
  released very quickly with an enzymes that
  degrades RNAs from 3-end inward.
• The figure 15.9 shows the requires poly(A) to be
  at the 3-end of the molecule.

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Functions of Ploy(A)

• 1.Protection of mRNA. The Figure 15.10 examined
  it.

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• 2.Translatability of mRNA. The Figure 15.11 shows
  effect of ployadenylation on translatability and
  stability of mRNA.

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• SUMMARY
• Transcription of eukaryotic genes extends
  beyond the polyadenylation site.Then the
  transcript is cleaved and polyadenylation at the
  3-end created by the cleavage.

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• Thank You !