Ch15 Posttranscriptional Events

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Ch15 Posttranscriptional Events
1. Capping Structure
-modification of 5-end of RNA
- 7-methylguanosine (m7G)
-occurs before chain length reaches 30 nt
Figure 15.4
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Cap 1 product
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Distribution of Cap structures
? Cap 1 found in both eukaryotic and viral
RNAs ? Cap 2 found only in eukaryotic cells ? Cap
0 (no 2-O methylated nucleotides) certain viral
RNAs
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• Functions of Caps
• Protection from degradation
• Improvement of translatability
• Transport of RNA (mRNA out of the nucleus)
• Proper splicing of the pre-mRNA

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Polyadenylation

• Modification of the 3 end ? addition of AMP
  chain
• -250 nt long, added posttranscriptionally by
  poly(A) polymerase
• -neither rRNA, tRNA, nor histone mRNAs contain
  poly(A) tails

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Functions of polyadenylation

• protection ? increases RNA lifetime
• translatability ? poly(A) binding protein I ?
  increases translational efficiency

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Overview of polyadenylation
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Transcription extends beyond polyadenylation site
? transcript is cleaved before polyadenylation
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Polyadenylation Signals

• An efficient mammalian polyadenylation signal
  consists of
• AAUAAA motif about 20 nt upstream of a
  polyadenylation site in a pre-mRNA
• Followed 23 or 24 bp later by GU-rich motif
• Followed immediately by a U-rich motif
• Variations on this theme occur in nature
• Results in variation in efficiency of
  polyadenylation
• Plant polyadenylation signals usually contain
  AAUAAA motif
• More variation exists in plant than in animal
  motif
• Yeast polyadenylation signals are even more
  different

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Cleavage of Pre-mRNA

• Polyadenylation involves both
• Pre-mRNA cleavage
• Polyadenylation at the cleavage site
• Cleavage in mammals requires several proteins
• CPSF cleavage and polyadenylation specificity
  factor
• CstF cleavage stimulation factor
• CF I
• CF II
• Poly (A) polymerase
• RNA polymerase II

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Initiation of Polyadenylation

• Short RNAs mimic a newly created mRNA 3-end can
  be polyadenylated
• Optimal signal for initiation of such
  polyadenylation of a cleaved substrate is AAUAAA
  followed by at least 8 nt
• When poly(A) reaches about 10 nt in length,
  further polyadenylation becomes independent of
  AAUAAA signal and depends on the poly(A) itself
• 2 proteins participate in the initiation process
• Poly(A)polymerase
• CPSF binds to the AAUAAA motif

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Elongation of Poly(A)

• Elongation of poly(A) in mammals requires a
  specificity factor called poly(A)-binding protein
  II (PAB II)
• This protein
• Binds to a preinitiated oligo(A)
• Aids poly(A) polymerase in elongating poly(A) to
  250 nt or more
• PAB II acts independently of AAUAAA motif
• Depends only on poly(A)
• Activity enhanced by CPSF

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• Initiation and elongation
• CPSF, CstF, CFI/II assemble on pre-mRNA guided by
  sequence motifs
• Cleavage stimulated by CTP of PolII CstF, CFI/II
  leave, PAP enters
• PAP (w/CPSF) initiates poly(A) synthesis (10 nt
  long)
• PAPII ?elongation

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• Poly(A) turnover, competition between RNAses and
  PAP ? poly(A) tail gone, mRNA degraded
• Polyadenylation can also occur in the cytoplasm
  requires both AAUAAA and upstream motif
  cytoplasmic polyadenylation element (CPE) UUUUUAU
• CAP Poly(A) effects on Splicing
• -removal of first intron dependent on CAP,
  removal of closest intron to poly(A) tail
  dependent on poly(A) tail