Outline

1
Outline

• Self-splicing RNAs
• Role of CTD in splicing
• Role of CAP in splicing
• Role of poly(A) in splicing

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Self-Splicing Introns

• Some Group I and Group II introns can self-splice
  in vitro in the absence of proteins (or other
  RNAs), i.e. they are ribozymes.
• Each group has a distinctive, semi-conserved
  secondary structure.
• Both groups require Mg2 to fold into a
  catalytically active ribozyme.
• Group I introns also require a guanosine
  nucleotide in the first step.

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Group II self splicing
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Tetrahymena rRNA Group I Intron

• First self-splicing intron was discovered by T.
  Cechs lab in 1981
• In the 26S rRNA gene in Tetrahymena (a protist)
• First example of a catalytic RNA!
• Nobel Prize in Chemistry to T. Cech and S. Altman
  (showed that RNase P was a true turnover
  riboenzyme in vivo), 1989

5
Group I splicing mechanism
GOH guanosine nucleotide guanosine will work
because the phosphates dont participate in the
reaction. In vivo, GTP probably used.
The 3 terminal G of the intron is nearly 100
conserved.
Fig. 14.47
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Cr.LSU intron 2ndary structure of a group I
intron
Old style drawing
Newer representation
Exon seq. in lower case and boxed
Shows how splice sites can be brought close
together by internal guide sequence.
Conserved core
5 splice site
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3-D Model of Tetrahymena rRNA Intron
Catalytic core consists of two stacked helices
domains 1. P5 P4 P6 P6a (in green) 2. P9
P7 P3 P8 (in purple) The substrate is
the P1 P10 domain (in red and black), it
contains both the 5 and 3 splice sites.
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Guanosine (G) binding-site of Group I Introns

• The incoming G becomes part of a triple helix
  with a G-C pair in P7of the conserved core.
• It is highly specific for Guanosine (Km for free
  GTP is 20 µM).
• Binds free GTP in the first splicing step.
• Binds the 3-terminal G of the intron in the
  second splicing step.

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Splicing Factors for Self-Splicing Introns

• Some Group I and many Group II introns cant
  self-splice in vitro (need protein factors?).
• Even self-splicing introns get help from proteins
  in vivo.
• First shown with fungal (yeast and Neurospora)
  mutants deficient in splicing of mitochondrial
  introns (respiratory-deficient).

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Protein splicing factors for Group I Introns

• 2 types
• Intron-encoded
• - promote splicing of the intron that encodes
  it
• Nuclear-encoded
• - for organellar introns

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Functions of nuclear-encoded splicing factors for
group I introns

• 1. Promote folding of the intron
• - promote tertiary structure
• - e.g., CBP2 promotes folding of a yeast
  cytochrome b intron
• 2. Stabilize correctly folded structure
• - Cyt18 (from Neurospora) promotes splicing of a
  number of group I introns
• - Cyt18 is also the mt tyrosyl-tRNA synthetase,
  dual-function protein
• - Evolved from a tyrosyl-tRNA synthetase by
  acquiring a new RNA-binding surface

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Remarkable similarities among three groups
13
Self splice and non-self splicing
Group II mitochondria
Colored bars are exons
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Figure 14.23
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RNAP II CTD

• Experiment CTD-GST stimulates splicing in vitro.
  GST does not.
• CTD binds snRNPs and splicing proteins.

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CTD-GST stimulates splicing in vitro
Figure 14.37
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Exon definition vs intron definition

• Intron definition is sufficient to identify ends
  of introns.
• For some transcripts the splicing machinery
  identifies the ends of introns without help from
  CTD.
• Exon definition is needed to successfully
  identify the ends of exons
• Here CTD helps to identify the ends of the EXONS.
• These types of transcripts are not splicing if
  the exons are not whole.

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Figure 14.39
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Cap stimulates splicing of the first intron in a
multi-intron pre-mRNA
32P-labeled substrate RNAs were incubated in a
Hela nuclear extract.
Splicing of 1st intron very poor with uncapped
pre-mRNA.
May have been methylation of Cap in extract.
Fig. 15.40
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CAP Binding Complex (CBP)

• Contains 2 proteins of 80 (CBP80) and 20 (CBP20)
  kiloDaltons
• Depletion of CBP from a splicing extract using
  antibody against CBP80 inhibited splicing of the
  first intron in a model pre-mRNA
• Further analysis showed an inhibition of
  spliceosome formation
• CBP may be important for spliceosome formation
  in vivo on first intron

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Poly A-Dependent Splicing of the Last Intron in a
2-intron pre-RNA
Splicing of the 2nd intron in this pre-mRNA is
reduced by a mutation in the polyadenylation
signal (WT hexamerAAUAAA). Splicing of the 1st
intron was normal.
Fig. 15.43
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Whats the origin of introns?
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Whats the advantage of introns?
-Alternative splicing to increase gene
diversity -Exon shuffling - -
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Discovery of Alternative Splicing

• First discovered with an Immunoglobulin heavy
  chain gene (D. Baltimore et al.)
• Alternative splicing gives two forms of the
  protein with different C-termini
• 1 form is shorter and secreted
• Other stays anchored in the plasma membrane via
  C-terminus

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Alternative splicing of the mouse immunoglobulin
µ heavy chain gene
S-signal peptide C - constant
region V- variable region green
membrane anchor Red- untranslated reg. yellow
end of coding reg. for secreted form
Fig. 14.37
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Regulation of Alternative splicing

• Sex determination in Drosophila involves 3
  regulatory genes that are differentially spliced
  in females versus males 2 of them affect
  alternative splicing
• Sxl (sex-lethal) - promotes alternative splicing
  of tra (exon 2 is skipped) and of its own (exon
  3 is skipped) pre-mRNA
• Tra promotes alternative splicing of dsx (last
  2 exons are excluded)
• Dsx (double-sex) - Alternatively spliced form of
  dsx needed to maintain female state

Fig. 14.38
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Alternative splicing in Drosophila maintains the
female state.
Alternative splicing
Sxl and Tra are SR proteins! Tra binds exon 4 in
dsx mRNA causing it to be retained in mature
mRNA.
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RNA Splicing and Disease

• 15 of the mutations that cause genetic
  diseases affect pre-mRNA splicing
• Many are cis-acting mutations at the
  splice-sites, the branch point, or sequences that
  promote (enhancers) or inhibit (silencers)
  splicing of certain exons