Messenger RNA Processing I: Splicing

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Messenger RNA Processing I Splicing

• Hui-Qiong Zhang

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(No Transcript)
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Spliceosome

• U1 is proved to pair with 5-splice sites and is
  essential for splicing.
• U6 might be involved in events near the 5-splice
  sites.
• U2 base pair withbranchpoint sequence
• U5 snRNA associates with the last nucleotide in
  one exon and the first nucleotide of the next,
  presumably lines the two exons up for splicing.
• U4 base-pairs with U6

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• Since the splicing occur,there exist a
  mechanism to determinate whether it should happen
  or not,thus comes out the concept commitment

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Commitment

• Some splicing factors play critical roles in
  commitment.
• Xiang-Dong Fu discovered in 1993 a single
  splicing factor, SC35 , can cause a commited
  complex to form.

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Commitment assay

• Splicing substratehuman ß-globin pre-mRNA
• Preincubation with purified SC35
• Add nuclear extract for 2 h
• Electrophoresis the RNAs

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Figure 14.31 Commitment of the human ß-globin
pre-mRNA
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SR proteins

• SC35 is a member of a group of RNA binding
  proteins called SR proteins
• SR proteins constitute the best-studied family
  ofsplicing regulators,which have common domain
  structures
• RBD(RNA binding domain)\RRM(RNA-recognition
  motif) bind RNA
• RS domian rich in serine (S) and arginine
  (R), mediates interactions between the SR protein
  and splicing machinery.
• .

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• Other SR proteins have commitment activity too?

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Figure 14.32 Commitment activities of several
RNA-binding proteins with the human ß-globin
pre-mRNA
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• Conclusion
• The commitment activity of SC35 is
  specific, and does not derive from a general
  RNA-binding capacity.

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Further proof of commitment pecificity

• Different splicing substrate
• HIV tat pre-mRNA
• (reported to be stimulated by SF2/ASF)

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Figure 14.33 Commitment activities of several
RNA-binding proteins with eitherß-globin pre-mRNA
or the HIV tat pre-mRNA
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Commitment of different pre-mRNAs requires
different splicing factors.
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Conclusion

• Commitment to splice at a given site is
  determined by an RNA-binding protein, which
  presumably binds to the splicing substrate and
  recruits other spliceosomal components, (starting
  with U1).

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Alternative Splicing

• Discussion of commitment leads to another
  important topic
• Alternative Splicing pre-mRNAs (about 1 in 20
  eukaryotic pre-mRNAs) can be spliced in more than
  one way, leading to two or more alternative mRNAs
  that encode different proteins.
• In human, 60 of transcripts are subjected to
  alternative splicing.

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Five ways to splice an RNA
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Example of alternative splicing

• In 1980 David Baltimore and colleagues discovered
  the first example of alternative splicing, the
  mouse immunoglobulin µheavy-chain gene
• A membranebound form µm
• hydrophobic region anchors it to the
  membrane
• A secreted form µs
• lack membrane anchor

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Experiments
clone germline gene
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Figure 14.40 Alternative splicing pattern in
the mouse immunoglobin µheavy-chain gene
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Drosophila sex determination

• Three different genes included in sex
  determination pathway
• Sex lethal Sxl
• Transformer tra
• Doublesex dsx

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Figure 14.41 Alternative splicing cascade in
Drosophila sex determination
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How is this alternative splicing controlled?
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• product of sxl and tra can determine which splice
  sites be used in tra and dsx transcripts
• these proteins are splicing factors that cause
  commitment
• In fact, the product of both sxl and tra
  are SR proteins

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Tra and Tra2 are necessary for alternative
splicing commitment, but are they sufficient ?

• Ming Tian and Maniatiss commitment assay
• labeled,shortened dsx pre-mRNA containing only
  exon3 and 4, intron in between
• micrococcal nuclease-treated nuclear extract
  (supply any proteins)
• electrophorese the RNAs and detect them by
  autoradiography

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Figure 14.42 Ability of various SR proteins to
complement Tra and Tra-2 in dsx splicing assay
1 no complementary Protein 2 a mixture of
SR Protein 16 highest amount of
recombination SC35 17-20 purified
Nonrecombination SR proteins
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• Tra and Tra-2 alone are not enough to cause
  commitment, however, something in the extract
  could complete them

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• To further clarify the mechanism of splice site
  selection, Maninatis and his colleagues focused
  on the female-specific splicing of dsx pre-mRNA
  by Tra and Tra-2, they discovered 300nt
  downstream of 3-splice site in dsx pre-mRNAwhich
  contains 6 repeats of a 13-nt sequence,known as
  repeat element
• Tra and Tra-2 bind to the repeat element

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• SR prteins bind to sequences within the
  exons (exonic splicing enhancers),from there
  recruit U2AF and U1snRNP to the downstream 5 and
  upstream 3splice sites respectively.

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Other splicing regulators
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Effects of alternative splicing
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Thank you!