Splicing of messenger RNAs

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Splicing of messenger RNAs
1. Historical context and background
2. The spliceosome, its reaction, and its assembly
3. Control of splice sites alternative splicing
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Splicing of messenger RNAs
1. Historical context and background
A. Discovery of introns and splicing
B. Splicing reaction
C. Signal sequences and enhancing elements (cap,
polyA)
2. The spliceosome, its reaction, and its assembly
3. Control of splice sites alternative splicing
3
Early Evidence For Splicing of mRNAs
Fig. 14.1
Discovered in adenovirus by Phil Sharp and
colleagues (1977)
EM of DNA from hexon gene (encoding viral coat
protein) hybridized to mature mRNA
Loops indicate introns regions present in DNA
but not RNA
Same method was used to demonstrate introns in
host mRNAs also, and to show that entire gene is
first transcribed, then spliced
Introns are also present in tRNA and rRNA
genes, and in organelles. We will focus here on
eukaryotic mRNAs.
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Basic Mechanism of Splicing
Fig. 14.2
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Chemical Reaction Mechanism of Pre-mRNA Splicing
Fig. 14.4
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Evidence For Splicing Intermediate
Gel electrophoresis of extract splicing
reaction demonstrates intermediate (1984)

• Intermediate and intron product shown to be
  lariat by
• anomalous migration
• digestion by nucleases and chemical treatment

Fig. 14.5
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Specific Sequences Define the Splice Sites
Mammalian consensus
5-AG/GUAAGU-intron-YNCURAC-YnNAG/G-3
Yeast consensus
5-/GUAUGU-intron-UACUAAC-YAG/-3
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Cap Formation and Poly(A) Addition Also Enhance
Splicing
Cap enhances splicing of first intron in HeLa
nuclear extract (Shimura, 1987)
Unmethylated cap shown to be methylated in
extract
Splicing enhancement later shown to depend on
formation of cap-binding complex (Mattaj and
colleagues, 1994)
Fig. 15.30
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Cap Formation and Poly(A) Addition Also Enhance
Splicing
Labeled RNAs added to nuclear extract
Polyadenylation signal allows poly(A) addition
in extract
Poly(A) addition affects splicing
No polyadenylation
Further work (not shown) revealed that effect
is only on the 3-most intron
Fig. 15.31
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Splicing of messenger RNAs
1. Historical context and background
2. The spliceosome, its reaction, and its assembly
A. Pieces of the spliceosome and interactions
with pre-mRNA
B. Is RNA the catalytic element of the
spliceosome?
C. Assembly and conformational transitions
3. Control of splice sites alternative splicing
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Splicing Is Carried Out by a Large Complex, the
Spliceosome
Spliceosome identified in yeast using glycerol
gradient ultracentrifugation with labeled
pre-mRNA (Brody and Abelson, 1985)
5 RNAs U1, U2, U4, U5, and U6 snRNAs, each in
a snRNP complex that includes proteins
More than 50 proteins total
Fig. 14.9
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Both RNA and Protein Components Interact With
Defined Regions of the Intron and Splice Sites
Fig. 14.10
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Structure of U1 snRNP
Cryo-EM of complex including - U1 snRNA -
Core Sm complex, 7 proteins - 3 U1 specific
proteins A, C, and 70K (only A and 70K are
visible)
Contacts with pre-mRNA made by 5-end of U1
snRNA, which protrudes from complex
Stark and Luhrmann, Ann. Rev. Biophys. Biomol.
Structure, 35 (2006), 435
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Spliceosome Structure
All snRNAs?
Abubel et al, Mol. Cell 15 (2004), 833-839
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Contacts Between U snRNAs and the pre-mRNA
Early base-pairing of pre-mRNA with U1 and U2
snRNA
Rearrangements allows base-pairing with U6 and
with U5 (not shown)
Protein contacts with pre-mRNA also
Hertel and Graveley, TIBS 30 (2005), 115-118
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U1 snRNA Base-pairs With the 5-Splice Site
Mutations in 5-splice site of adenovirus gene
and compensating changes in U1 snRNA
Fig. 14.12
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U6 snRNA Can Also Base-pair With the 5-Splice
Site
Various approaches, particularly
photocrosslinking, showed interaction of U6 with
5-splice site
Reversible crosslinking with psoralen was
particularly informative (on next
slide) Sontheimer and Steitz, 1992
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Interactions Detected By Reversible Psoralen
Cross-linking and 2D Electrophoresis
Complexes isolated by antibody to U snRNA cap
and biotin from pre-mRNA
Crosslinks reversed before gel run in second
dimension
Fig. 14.16
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U2 snRNA Forms Base Pairs With the Branchpoint
Sequence
Mutations in branchpoint sequence abolished
splicing, and compensating changes in U2
rescued splicing
Guthrie and colleagues used genetic assay
failure to splice did not allow yeast to grow on
media containing histidine precursor histidinol
Fig. 14.17
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U5 snRNA Associates With Both Splice Sites
Crosslinking by 4-thio-U at first position of
second exon
Identity of crosslinked RNA determined by RNase
H cleavage
Other experiments established contact with
first exon
Fig. 14.21
Fig. 14.19
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Catalytic RNA In the Spliceosome?
Some similarity between spliceosome and group
II intron, which is autocatalytic
Fig. 14.22
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Further Support For RNA Catalysis In the
Spliceosome
 In-vitro-synthesized U2 and U6 snRNA can
catalyze a reaction related to the 1st step when
added to an intron fragment (Valadkhan and
Manley, 2001)
It remains unclear whether these reactions
truly mimic splicing reactions
Fig. 14.23
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Assembly of the Spliceosome
RNA immobilized to beads through biotin-avidin
linkage of complementary oligonucleotide
U1 appears to associate first in Northern blot
analysis
Fig. 14.25
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Step-wise Spliceosome Assembly
Wahl et al, Cell 136 (2009), 701-718
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Does the Spliceosome Come Pre-assembled?
Gentle extraction procedures revealed large,
catalytic complex
Stevens et al, Mol Cell 9 (2002), 31-44.
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Conformational Changes Require DExD/H-box
Remodelers
Extensive rearrangements are necessary to
generate correct base pairing for splicing
Several ATP-dependent steps in splicing. All
ATP hydrolysis is used for conformational
rearrangements, none for energetics of the
chemical reactions
Wahl et al, Cell 136 (2009), 701-718
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Extensive Base-pairing Changes During Spliceosome
Activation
Most dramatic example is necessity for disruption
of pairing between U6 and U4
Staley and Guthrie, Cell 92 (1998), 315-326
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Splicing of messenger RNAs
1. Historical context and background
2. The spliceosome, its reaction, and its assembly
3. Control of splice sites alternative splicing
A. Types of alternative splicing
B. SR proteins as major splicing regulators
C. Examples of alternative splicing
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Types of Alternative Splicing
Alternative 5-ss
Alternative 3-ss
Exon inclusion/exclusion
Modular exons
Intron inclusion/exclusion
Smith and Valcarcel, TIBS 25 (2000), 381-388
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SR Proteins Key Mediators of Alternative and
Constitutive Splicing Events in Animals
RRMRNA Recognition Motif RSArg/Ser-rich
domain
RS
RRM
binds to pre-mRNA sequence protein/RNA
interaction domain
SR proteins function in both constitutive and
alternative splicing
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Postulated Roles of SR Proteins in Constitutive
Splicing
ESEexonic splicing enhancers
For long introns, spliceosomal first assemble
around exons, not introns
U1 recognizes downstream 5-splice site, U2AF
recognizes upstream 3-splice site, and SR
proteins form cross-exon interactions
These cross-exon interactions are later
replaced by cross-intron interactions in a series
of events that are not well understood
Wahl et al, Cell 136 (2009), 701-718
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An Alternative Splicing Cascade In Sex
Determination in Drosophila
Alternative splicing gives active proteins in
females, inactive proteins in males
Optional exon
Alternative 3-ss
Alternative 3-ss
Fig. 14.39
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The Insect Dscam Gene Can Code For 38,016
Different Protein Isoforms
Graveley, Cell 123 (2005), 65-73
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Proposed Mechanism For Alternative Dscam Splicing
Graveley, Cell 123 (2005), 65-73
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Key Points
1. Pre-mRNA splicing proceeds in two steps.
First, the 2-OH from an A within the intron
attacks at the 5-splice site to give a lariat
intermediate. Second, the 3-OH from the 5-exon
attacks the 3-splice site to generate ligated
exons and excised lariat intron.
2. Splicing is carried out by the spliceosome 5
RNAs and many proteins. It assembles and
rearranges in complex process.
3. Many genes undergo alternative splicing.
Splice site choice is influenced by SR proteins.
Chosen sites are frequently determined by exon
definition, using exonic splicing enhancer
sequences.
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