Outline

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Outline

• Splicing

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RNA Splicing

• RNA splicing is the removal of intervening
  sequences (IVS) that interrupt the coding region
  of a gene
• Excision of the IVS (intron) is accompanied by
  the precise ligation of the coding regions (exons)

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Discovery of Split Genes (1977)

• P. Sharp and R. Roberts - 1993 Nobel Prize in
  Physiology Medicine
• Discovered using R-loop Analysis
• Cloned genomic DNAs of a few highly expressed
  nuclear genes (e.g., hemoglobin, ovalbumin), and
  certain Adenoviral genes were hybridized to RNA
  fractions and visualized by EM
• Loops form from RNA annealing to the template
  strand and displacing coding strand of DNA

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Figure 14.3
mouse globin mRNA PRECURSOR RNA hybridized to
cloned gene (genomic).
mouse globin MATURE mRNA hybridized to cloned
gene (genomic).
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Intron Classes Distribution

• Group I - common in organelles, nuclear rRNA
  genes of lower eukaryotes, a few prokaryotes
  (gt1500 known)
• Group II - common in organelles, also in some
  prokaryotes and archaea (gt 1000)
• Nuclear mRNA (NmRNA) - ubiquitous in eucaryotes
• Nuclear tRNA- some eucaryotes

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Relationships of the 4 Intron Classes

• Each class has a distinctive structure
• The chemistry of splicing of Groups I, II and
  NmRNA is similar i.e, transesterification
  reactions
• The splicing pathway for Group II and nuclear
  mRNA introns are similar
• Splicing of Groups I, II and possibly NmRNA
  introns are RNA-catalyzed

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Structure of NmRNA Introns

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

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Splicing Signals
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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. 14.9
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Figure 14.4
Mechanisms of Splicing two step
transesterification
Fig. 14.4
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• Weaver pg 429
• Sharp coworkers
• intron is 231n
• DO is an antiSNURP sera
• ME is the control sera

4 poly- acrylamide gel 8M urea
Very hard to see band
10 poly- acrylamide gel 8 M urea
Fig. 14.5
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• Weaver pg 429
• Sharp coworkers
• intron is 231n
• DO is an antiSNURP sera
• ME is the control sera
• Panel C left out of textbook. You cant properly
  interpret it without panel C. It is a Southern
  blot of the 10 gel probed with a L1L2 fragment.

4 poly- acrylamide gel 8M urea
Very hard to see band
10 poly- acrylamide gel 8 M urea
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Time course of intermediate and liberated intron
appearance
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.
Fig. 14.6
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What is the branchpoint nucleotide?Is the
branchpoint attached to 5 or 3 end?
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TLC to demonstrate that A is the branchpoint
Fig. 14.8
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Is the branched nucleotide attached to the 5 end
of the intron?
RNase T1 cuts after guanylate residues. RNase T1
cuts only single stranded RNA.
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Figure 14.10
Splicing occurs in spliceosome
Yeast spliceosome 40S Mammalian 60S
Blue has a mutation in A to C
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Spliceosomes contain Snurps (a.k.a., snRNPs or
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 some with each other (U4-U6 pair in
  snurps, and U2-U6 pair in spliceosome).
• Lupus patients have antibodies to snurps mainly
  the Sm proteins.

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Roles of snRNAs/Snurps

• U1 base-pairs with the 5 splice-site
• U2 binds/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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3 splice site also called the acceptor site
5 splice site also called the donor site
What is the evidence that the Snurps are
necessary for splicing?
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Fig. 14.11
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Figure 14.12 14.13 pg 434
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Figure 14.12 14.13 pg 434
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Figure 14.12 14.13 pg 434
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Figure 14.12 14.13 pg 434
Base pairing is requried but is it sufficient?
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Figure 14.12 14.13 pg 434
Base pairing is requried but is it sufficient?
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Figure 14.12 14.13 pg 434
Base pairing is requried but is it sufficient?
Clearly base pairing with U1 is not all that is
required.
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Spliceosome Cycle
Fig 14.28
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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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snRNAs

• U1 - recognition (bp) of the 5 splice site
  (donor site).
• U2 - branch point recognition (bp) bps to U6
  snRNA
• U5 binds ends of exons
• U4 binds U6 and holds it untile U6 is needed in a
  splicing reaction
• U6 - bps to 5 splice site and U2 snRNA U4
  snRNA
• 3 splice site should be 18-40n downstream of
  branch point. Slu7 and U2AF use branch point to
  help recognize 3 splice site.

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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 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 near the 3 end of the intron
• Helps define the intron prior to splicing