6 domains,

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Consensus Group II intron Structure
6 domains, Helical Wheel Domain I contains
binding sites for the 5 exon (keeps the 5 exon
from floating away after the first splicing step)
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Group II Splicing Pathway
(1) The 2 OH of a special internal A attacks the
5 splice site creating a branched intron
structure.
(2) The 3 OH of the 5 exon attacks the 3
splice site, ligating the exons and releasing the
intron as a lariat structure.
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Structure of NmRNA Introns

• Most begin with GU and end with AG.
• Most of the internal sequences are not
  conserved.
• However, there are other important consensus
  sequences near the ends (in addition to GU and
  AG).

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Consensus Mammalian NmRNA Splice Signals
5 ag/GUAAGU -------YNCURAC---YnNAG/g 3
Y - pyrimidine (U or C) Yn - string of 9
pyrimidines R - purine (A or G) N - any base
Branch site sequence
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Nm RNA Splicing MechanismHow it has been
studied.

• Elucidating the overall mechanism, cis elements,
  and trans factors depended on
• Site-directed mutagenesis of genes in vitro, and
  subsequent expression in vivo (yeast, Hela
  cells, and others).
• Development of accurate splicing extracts (HeLa
  cells and yeast).
• Isolation of temperature-sensitive yeast mutants
  defective in NmRNA splicing.

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Kinetics of In vitro Splicing in a Hela cell
nuclear extract
Exon 2
Pre - radioactively labeled precursor RNA
- The splicing reactions were separated by gel
electrophoresis. Notice that the intron and
intron-exon RNAs have an unusually reduced
mobility in these polyacrylamide-urea
gels. There is also some cleavage at the Exon 2
- Intron 2 splice-site, producing the Spliced
Exons molecule.
Spliced exons
Fig. 14.5
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Plot of changes in amounts of products and
intermediates during the splicing reaction in the
previous slide.
Fig. 14.5
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2 Transesterification reactions w/phosphates
1st 2OH of internal A attacks the
phosphodiester that links Exon 1 to the 5 end of
the intron (producing lariat and 5 exon
molecules) 2nd - 3OH of the 5 exon attacks
the phosphodiester that links Exon 2 to the 3
end of the intron ( ligating the exons) No. of
phosphodiesters is conserved!
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In yeast, the branch-point sequence determines
which downstream AG is used.
Exon 1
Exon 2
branch
Inserted sequence
Branch site moved into exon 2.
Branch sequence
RNAs that were tested for splicing in vivo.
Fig. 14.8
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NmRNA Splicing occurs on Spliceosomes!
Strategy An Adenovirus pre-mRNA (32P-labeled)
was incubated in a HeLa cell nuclear extract to
allow splicing to begin. Then the extract was
centrifuged down a glycerol gradient to size the
complexes that formed.
Result The intron-exon 2 intermediate sedimented
at 60S on a glycerol gradient much bigger than
expected for naked RNA.
Question What else is in the 60S spliceosome
complex?
Fig. 14.13, 2ed.
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Spliceosomes contain Snurps (snRNPs, small
nuclear ribonucleoproteins)

• A snurp contains a small, nuclear, U-rich RNA
  (snRNAs U1, U2, U4, U5 or U6), and gt 7
  proteins, 5 (Sm) are common.
• The snRNAs base-pair with the pre-mRNA (U1, U2,
  U5, U6) and/or with each other (U4-U6 and U2-U6).
  
• Lupus patients have antibodies to snurps mainly
  the Sm proteins.

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Fig. 14.28
Structure of the U1 SnRNP
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Roles of snRNAs/Snurps

• U1 pairs with the 5 splice-site.
• U2 pairs with the branch point also pairs with
  U6 in the assembled spliceosome.
• U4 pairs with U6 in SnRNPs, but unpairs during
  spliceosome assembly.
• U5 interacts with both exons (only 1-2 nt
  adjacent to intron) helps bring exons together.
  
• U6 displaces U1 at the 5 splice-site (pairs with
  nt in the intron) it also pairs with U2 in the
  catalytic center of the spliceosome.

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U1 and U2 paired with pre-mRNA in yeast
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Similar active sites (catalytic center) in
Spliceosomal and Group II introns?
(both models after first step)
Fig. 14.22
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The Spliceosome Cycle of Assembly, Rxn, and
Disassembly
Fig. 14.27
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Intermediate complexes in the Spliceosome cycle

• CC is the commitment complex (contains U1 on
  the pre-mRNA)
• A also contains U2
• B1 also contains U6-U4/U5
• B2 lacks U1 and U4, activated spliceosome
• C1 contains 5-exon intron-exon
• C2 contains intron-lariat and ligated exons

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Some Unique Features of the Spliceosome

• Transient complex that forms on pre-mRNA.
  Contrast with ribosomal subunits, which are
  completely stable.
• Ribonucleoprotein components of the spliceosome,
  snurps, are stable structures.
• In yeast, the spliceosome sediments at 40S
  whereas in humans it is 60S (ribosomal subunits
  from these species are similar in size).

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Proteins that promote formation of the Commitment
Complex

• In humans the SR proteins SC35 and SF2 commit
  splicing on globin HIV Tat pre-mRNA
• SR proteins have domains rich in serine and
  arginine
• In yeast the branch-point bridging protein (BBP)
  binds to the U1 snurp at the 5 end of the
  intron, and RNA and Mud2p near the 3 end of the
  intron
• Helps define the intron prior to splicing

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Figure 14.36
Fig. 14.34a
SS - splice site BP - branch point

Branch-point Bridging Protein (BBP) binds RNA
(near the 3 end of intron) and 2 proteins (U1
SnRNP Mud2p). Helps define the intron portion.