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Ch15 Posttranscriptional Events

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Cap 1 found in both eukaryotic and viral RNAs. Cap 2 found only in eukaryotic cells ... More variation exists in plant than in animal motif ... – PowerPoint PPT presentation

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Title: Ch15 Posttranscriptional Events


1
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
2
Cap 1 product
3
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4
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
5
  • Functions of Caps
  • Protection from degradation
  • Improvement of translatability
  • Transport of RNA (mRNA out of the nucleus)
  • Proper splicing of the pre-mRNA

6
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

7
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8
Functions of polyadenylation
  • protection ? increases RNA lifetime
  • translatability ? poly(A) binding protein I ?
    increases translational efficiency

9
Overview of polyadenylation
10
Transcription extends beyond polyadenylation site
? transcript is cleaved before polyadenylation
11
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

12
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

13
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

14
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

15
  • 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

16
  • 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
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