Welcome Each
of You to My
Molecular
Biology Class
Molecular Biology of the
Gene,5/E --- Watson et al,
(2004)
Part I,Chemistry and Genetics
Part II,Maintenance of the
Genome
Part III,Expression of the
Genome
Part IV,Regulation
Part V,Methods
4/3/05
Ch 12, Mechanisms of
Transcription
Ch 13, RNA Splicing
Ch 14, Translation
Ch 15, The Genetic code
4/3/05
CHAPTER 13
RNA Splicing
?Molecular Biology Course
Figure 13-1
Primary transcript
Most of the eukaryotic genes are
mosaic (嵌合体 ),consisting of
intervening sequences separating the
coding sequence
? Exons (外显子 ),the coding sequences
? Introns (内含子 ), the intervening
sequences
? RNA splicing,the process by which
introns are removed from the pre-
mRNA.
? Alternative splicing (可变剪接 ),some
pre-mRNAs can be spliced in more
than one way,generating alternative
mRNAs,60% of the human genes are
spliced in this manner.
Topic 1, THE
CHEMISTRY OF RNA
SPLICING
CHAPTER 13 RNA Splicing
Sequences within the RNA
Determine Where Splicing
Occurs
Th
e
ch
em
ist
ry
of
RN
A
sp
lic
in
g
The borders between introns
and exons are marked by
specific nucleotide sequences
within the pre-mRNAs.
Figure 13-2
The consensus sequences for human
? 5’splice site (5’剪接位点 ),the exon-
intron boundary at the 5’ end of
the intron
? 3’ splice site (3’剪接位点 ),the
exon-intron boundary at the 3’
end of the intron
? Branch point site (分枝位点 ),an A
close to the 3’ end of the intron,
which is followed by a
polypyrimidine tract (Py tract).
The intron is removed in a
Form Called a Lariat (套马索 ) as
the Flanking Exons are joined
Two successive transesterification:
Step 1,The OH of the conserved A
at the branch site attacks the
phosphoryl group of the
conserved G in the 5’ splice site,
As a result,the 5’ exon is
released and the 5’-end of the
intron forms a three-way
junction structure.
Th
e
ch
em
ist
ry
of
RN
A
sp
lic
in
g
Figure 13-3
Three-way
junction
The structure of three-way
function
Figure 13-4
Step 2,The OH of the 5’ exon
attacks the phosphoryl group
at the 3’ splice site,As a
consequence,the 5’ and 3’
exons are joined and the
intron is liberated in the
shape of a lariat.
Figure 13-3
Exons from different RNA
molecules can be fused by
Trans-splicing
? Trans-splicing,the process in
which two exons carried on
different RNA molecules can be
spliced together,
Th
e
ch
em
ist
ry
of
RN
A
sp
lic
in
g
Trans-splicing
Figure 13-5
Not a lariat
Topic 2
THE SPLICESOME
MACHINERY
CHAPTER 13 RNA Splicing
RNA splicing is carried out by a
large complex called spliceosome
? The above described splicing of
introns from pre-mRNA are
mediated by the spliceosome.
? The spliceosome comprises
about 150 proteins and 5
snRNAs (?).
? Many functions of the
spliceosome are carried out by
its RNA components.
Th
e
sp
lic
eo
so
m
e
m
ac
hi
ne
ry
? The five RNAs (U1,U2,U4,U5,and
U6,100-300 nt) are called small
nuclear RNAs (snRNAs).
? The complexes of snRNA and
proteins are called small nuclear
ribonuclear proteins (snRNP,
pronounces,snurps”).
? The spliceosome is the largest
snRNP,and the exact makeup
differs at different stages of the
splicing reaction
? Three roles of snRNPs in splicing
1,Recognizing the 5’ splice site
and the branch site.
2,Bringing those sites together.
3,Catalyzing (or helping to
catalyze) the RNA cleavage.
RNA-RNA,RNA-protein and
protein-protein interactions are
all important during splicing.
Figure 13-6
RNA-RNA interactions between different
snRNPs,and between snRNPs and pre-mRNA
Topic 3
SPLICING
PATHWAYS
CHAPTER 13 RNA Splicing
Assembly,rearrangement,and
catalysis within the spliceosome,
the splicing pathway (Fig,13-8)
? Assembly step 1
1,U1 recognize 5’ splice site,
2,One subunit of U2AF binds to
Py tract and the other to the 3’
splice site,The former subunits
interacts with BBP and helps it
bind to the branch point.
3,Early (E) complex is formed
Sp
lic
in
g
pa
th
w
ays
? Assembly step 2
1,U2 binds to the branch site,and
then A complex is formed.
2,The base-pairing between the
U2 and the branch site is such
that the branch site A is
extruded(Figure 13-6),This A
residue is available to react
with the 5’ splice site.
Figure 13-8
E complex
A complex
Figure 13-6b
? Assembly step 3
1,U4,U5 and U6 form the tri-snRNP
Particle,
2,With the entry of the tri-snRNP,
the A complex is converted into
the B complex.
Figure 13-8
A complex
B complex
? Assembly step 4
U1 leaves the complex,and U6
replaces it at the 5’ splice site.
U4 is released from the complex,
allowing U6 to interact with U2
(Figure 13-6c).This arrangement
called the C complex.
Figure 13-8Figure 13-6c
B complex
C complex
in which the
catalysis has
not occurred
yet
Catalysis Step 1:
Formation of the C complex
produces the active site,with U2
and U6 RNAs being brought
together
Formation of the active site
juxtaposes the 5’ splice site of
the pre-mRNA and the branch
site,allowing the branched A
residue to attack the 5’ splice
site to accomplish the first
transesterfication reaction.
Catalysis Step 2:
U5 snRNP helps to bring the two
exons together,and aids the
second transesterification
reaction,in which the 3’-OH of
the 5’ exon attacks the 3’ splice
site.
? Final Step:
Release of the mRNA product
and the snRNPs
Figure 13-8
C complex
E complex
A complex
B complex
C complex （没有
该 complex的图）
splicesome-mediated splicing reactions
Figure 13-8
Self-splicing introns reveal that
RNA can catalyze RNA splicing
? Self-splicing introns,the intron
itself folds into a specific
conformation within the
precursor RNA and catalyzes
the chemistry of its own release
and the exon ligation
Sp
lic
in
g
pa
th
w
ays
Adams et al.,Nature 2004,Crystal structure of a
self-splicing group I intron with both exons
Practical definition for self-splicing
introns,the introns that can remove
themselves from pre-RNAs in the
test tube in the absence of any
proteins or other RNAs,
There are two classes of self-
splicing introns,group I and group
II self-splicing introns.
TABLE 13-1 Three class of RNA Splicing
Class Abundance Mechanism Catalytic
Machinery
Nuclear
pre-
mRNA
Very common; used
for most eukaryotic
genes
Two
transesterificat
ion reactions;
branch site A
Major
spliceosom
e
Group
II
introns
Rare; some eu-
Karyotic genes from
organelles and
prokaryotes
Same as pre-
mRNA
RNA
enzyme
encoded
by intron
(ribozyme)
Group I
introns
Rare; nuclear rRNA in
some eukaryotics,
organlle genes,and a
few prokaryotic genes
Two
transesterific-
ation reactions;
exogenous G
Same as
group II
introns
? The chemistry of group II
intron splicing and RNA
intermediates produced are
the same as that of the
nuclear pre-mRNA.
Figure 13-9
Group I introns release a linear
intron rather than a lariat
? Instead of using a branch point A,
group I introns use a free G to attack
the 5’ splice site.
? This G is attached to the 5’ end of the
intron.The 3’-OH group of the 5’ exon
attacks the 5’ splice site.
? The two-step transesterification
reactions are the same as that of
splicing of the group II intron and
pre-mRNA introns.
Sp
lic
in
g
pa
th
w
ays
G instead of A
a linear introna Lariat intron
Figure 13-9
1,Smaller than group II introns
2,Share a conserved secondary
structure,which includes an
“internal guide sequence” base-
pairing with the 5’ splice site
sequence in the upstream exon.
3,The tertiary structure contains a
binding pocket that will
accommodate the guanine
nucleotide or nucleoside cofactor
Group I introns
The similarity of the structures
of group II introns and U2-U6
snRNA complex formed to
process first transesterification
Figure
13-10
How does spliceosome find
the splice sites reliably
Sp
lic
in
g
pa
th
w
ays
Two kinds of splice-site
recognition errors
? Splice sites can be skipped.
?,Pseudo” splice sites could be
mistakenly recognized,
particularly the 3’ splice site,
Figure 13-12
Reasons for the recognition
errors
(1) The average exon is 150 nt,
and the average intron is about
3,000 nt long (some introns are
near 800,000 nt)
? It is quite challenging for the
spliceosome to identify the
exons within a vast ocean of the
intronic sequences,
(2) The splice site consensus
sequence are rather loose,For
example,only AG?G tri-
nucleotides is required for the 3’
splice site,and this consensus
sequence occurs every 64 nt
theoretically,
1,Because the C-terminal tail of
the RNA polymerase II carries
various splicing proteins,co-
transcriptional loading of these
proteins to the newly synthesized
RNA ensures all the splice sites
emerging from RNAP II are
readily recognized,thus
preventing exon skipping,
Two ways to enhance the
accuracy of the splice-site
selection
2,There is a mechanism to
ensure that the splice sites close
to exons are recognized
preferentially,SR proteins bind
to the ESEs (exonic splicing
enhancers) present in the exons
and promote the use of the
nearby splice sites by recruiting
the splicing machinery to those
sites
SR proteins,bound to exonic
splicing enhancers (ESEs),
interact with components of
splicing machinery,recruiting
them to the nearby splice sites,
Figure 13-13
1,Ensure the accuracy and
efficacy of constitutive splicing
2,Regulate alternative splicing
3,There are many varieties of SR
proteins,Some are expressed
preferentially in certain cell
types and control splicing in
cell-type specific patterns
SR proteins are essential for
splicing
Topic 4
ALTERNATIVE
SPLICING
CHAPTER 13 RNA Splicing
? Many genes in higher eukaryotes
encode RNAs that can be spliced
in alternative ways to generate
two or more different mRNAs
and,thus,different protein
products.
Single genes can produce multiple
products by alternative splicing
Al
te
rn
at
ive
sp
lic
in
g
Drosophila DSCAM gene
can be spliced in 38,000
alternative ways
Figure 13-13
Figure 13-15
There are five different ways to
alternatively splice a pre-mRNA
Alternative splicing can be either
constitutive or regulated
? Constitutive alternative splicing,
more than one product is always
made from a pre-mRNA
? Regulative alternative splicing,
different forms of mRNA are
produced at different time,
under different conditions,or in
different cell or tissue types
An example of constitutive
alternative splicing, Splicing of
the SV40 T antigen RNA
Figure 13-16
Alternative splicing is regulated
by activators and repressors
? The regulating sequences,
exonic (or intronic) splicing
enhancers (ESE or ISE) or
silencers (ESS and ISS),The
former enhance and the latter
repress splicing.
? Proteins that regulate splicing
bind to these specific sites for
their action
Al
te
rn
at
ive
sp
lic
in
g
? SR proteins binding to enhancers
act as activators.
(1) One domain is the RNA-
recognition motif (RRM)
(2) The other domain is RS domain
rich in arginine and serine,This
domain mediates interactions
between the SR proteins and
proteins within the splicing
machinery.
? hnRNPs binds RNA and act as
repressors
1,Most silencers are recognized by
hnRNP ( heterogeneous nuclear
ribonucleoprotein) family,
2,These proteins bind RNA,but
lack the RS domains,Therefore,
(1) They cannot recruit the
splicing machinery,(2) they
block the use of the specific
splice sites that they bind.
Regulated alternative splicing
Figure 13-17
Binds at each end
of the exon and
conceals (隐藏 ) it
Coats the RNA and
makes the exons
invisible to the
splicing machinery
An example of repressors,
inhibition of splicing by hnRNPI
Figure 13-18
? The outcome of alternative
splicing:
1,Producing multiple protein
products,called isoforms.
2,Switching on and off the
expression of a given gene,In
this case,one functional
protein is produced by a
splicing pattern,and the non-
functional proteins are resulted
from other splicing patterns,
A small group of intron are
spliced by minor spliceosome
? This spliceosome works on
a minority of exons,and
those have distinct splice-
site sequence,
? The chemical pathway is the
same as the major
spliceosome.
Al
te
rn
at
ive
sp
lic
in
g
Figure 13-19
The AT-AC
spliceosome
U11 and U12 are in
places of U1 and U2,
respectively
Topic 5
EXON
SHUFFLING
CHAPTER 13 RNA Splicing
? All eukaryotes have introns,and
yet these elements are rare in
bacteria,Two likely explanations
for these situation,
1,Introns early model – introns
existed in all organisms but have
been lost from bacteria.
2,Intron late model – introns
never existed in bacteria but
rather arose later in evolution.
Ex
on
sh
uf
fli
ng
Exons are shuffled by recombin-
ation to produce gene encoding
new proteins
? Why have the introns been
retained in eukaryotes?
1,The need to remove introns,
allows for alternative splicing
which can generate multiple
proteins from a single gene,
2,Having the coding sequence of
genes divided into several
exons allows new genes to be
created by reshuffling exon.
? Three observations suggest exon
shuffling actually occur:
1,The borders between exons and
introns within a gene often
coincide with the boundaries
between domains within the
protein encoded by that gene.
For example,DNA-binding protein
Figure 13-21
2,Many genes,and proteins they
encode,have apparently arisen
during evolution in part via exon
duplication and divergence.
3,Related exons are sometimes
found in unrelated genes.
Exons have been reused in genes
encoding different proteins
Figure 13-22
Topic 6
RNA
EDITING
CHAPTER 13 RNA Splicing
RNA editing is another way
of changing the sequence of
an mRNA
I,Site specific deamination,
1,A specifically targeted C
residue within mRNA is
converted into U by the
deaminase.
2,The process occurs only in
certain tissues or cell types
and in a regulated manner.
RN
A
ed
iti
ng
Figure 13-25
The human
apolipoprotein gene
Stop code
In liver In intestines
Figure 13-25
3,Adenosine deamination also
occurs in cells,The enzyme
ADAR (adenosine deaminase
acting on RNA) convert A into
Inosine,Insone can base-pair
with C,and this change can alter
the sequence of the protein,
4,An ion channel expressed in
mammalian brains is the target
of Adenosine deamination.
II Guide RNA-directed uridine
insertion or deletion.
1,This form of RNA editing is
found in the mitochondria of
trypanosomes.
2,Multiple Us are inserted into
specific region of mRNAs after
transcription (or US may be
deleted).
3,The addition of Us to the
message changes codons and
reading frames,completely
altering the,meaning” of the
message.
4,Us are inserted into the message
by guide RNAs (gRNAs),
Having three regions,
anchor– directing the gRNAs to
the region of mRNAs it will edit.
editing region – determining
where the Us will be inserted
poly-U stretch
gRNAs
Figure 13-26
Topic 7
mRNA
TRANSPORT
CHAPTER 13 RNA Splicing
Once processed,mRNA is packaged
and exported from the nucleus into
the cytoplasm for translation
m
RN
A
tr
an
sp
or
t
? All the fully processed mRNAs
are transported to the
cytoplasm for translation into
proteins
? Movement from the nucleus to the
cytoplasm is an active and carefully
regulated process.
? The damaged,misprocessed and
liberated introns are retained in the
nucleus and degraded.
1.A typical mature mRNA carries a
collection of proteins that identifies
it as being ready for transport,
2.Export takes place through the
nuclear pore complex,
3.Once in the cytoplasm,some
proteins are discarded and are
then imported back to the
nucleus for another cycle of
mRNA transport,Some proteins
stay on the mRNA to facilitate
translation.
Figure 13-27
1,Why RNA splicing is important?
2,Chemical reaction,determination of the
splice sites,the products,trans-splicing
3,Spliceosome,splicing pathway and finding
the splice sites
4,Self-splicing introns and mechanisms
5,Alternative splicing and regulation,
alternative spliceosome
6,Two different mechanisms of RNA editing
7,mRNA transport-a link to translation
Key points of the chapter
Watch the animations
Enjoy the critical
thinking excises on
your study CD,
HOMEWORKS
of You to My
Molecular
Biology Class
Molecular Biology of the
Gene,5/E --- Watson et al,
(2004)
Part I,Chemistry and Genetics
Part II,Maintenance of the
Genome
Part III,Expression of the
Genome
Part IV,Regulation
Part V,Methods
4/3/05
Ch 12, Mechanisms of
Transcription
Ch 13, RNA Splicing
Ch 14, Translation
Ch 15, The Genetic code
4/3/05
CHAPTER 13
RNA Splicing
?Molecular Biology Course
Figure 13-1
Primary transcript
Most of the eukaryotic genes are
mosaic (嵌合体 ),consisting of
intervening sequences separating the
coding sequence
? Exons (外显子 ),the coding sequences
? Introns (内含子 ), the intervening
sequences
? RNA splicing,the process by which
introns are removed from the pre-
mRNA.
? Alternative splicing (可变剪接 ),some
pre-mRNAs can be spliced in more
than one way,generating alternative
mRNAs,60% of the human genes are
spliced in this manner.
Topic 1, THE
CHEMISTRY OF RNA
SPLICING
CHAPTER 13 RNA Splicing
Sequences within the RNA
Determine Where Splicing
Occurs
Th
e
ch
em
ist
ry
of
RN
A
sp
lic
in
g
The borders between introns
and exons are marked by
specific nucleotide sequences
within the pre-mRNAs.
Figure 13-2
The consensus sequences for human
? 5’splice site (5’剪接位点 ),the exon-
intron boundary at the 5’ end of
the intron
? 3’ splice site (3’剪接位点 ),the
exon-intron boundary at the 3’
end of the intron
? Branch point site (分枝位点 ),an A
close to the 3’ end of the intron,
which is followed by a
polypyrimidine tract (Py tract).
The intron is removed in a
Form Called a Lariat (套马索 ) as
the Flanking Exons are joined
Two successive transesterification:
Step 1,The OH of the conserved A
at the branch site attacks the
phosphoryl group of the
conserved G in the 5’ splice site,
As a result,the 5’ exon is
released and the 5’-end of the
intron forms a three-way
junction structure.
Th
e
ch
em
ist
ry
of
RN
A
sp
lic
in
g
Figure 13-3
Three-way
junction
The structure of three-way
function
Figure 13-4
Step 2,The OH of the 5’ exon
attacks the phosphoryl group
at the 3’ splice site,As a
consequence,the 5’ and 3’
exons are joined and the
intron is liberated in the
shape of a lariat.
Figure 13-3
Exons from different RNA
molecules can be fused by
Trans-splicing
? Trans-splicing,the process in
which two exons carried on
different RNA molecules can be
spliced together,
Th
e
ch
em
ist
ry
of
RN
A
sp
lic
in
g
Trans-splicing
Figure 13-5
Not a lariat
Topic 2
THE SPLICESOME
MACHINERY
CHAPTER 13 RNA Splicing
RNA splicing is carried out by a
large complex called spliceosome
? The above described splicing of
introns from pre-mRNA are
mediated by the spliceosome.
? The spliceosome comprises
about 150 proteins and 5
snRNAs (?).
? Many functions of the
spliceosome are carried out by
its RNA components.
Th
e
sp
lic
eo
so
m
e
m
ac
hi
ne
ry
? The five RNAs (U1,U2,U4,U5,and
U6,100-300 nt) are called small
nuclear RNAs (snRNAs).
? The complexes of snRNA and
proteins are called small nuclear
ribonuclear proteins (snRNP,
pronounces,snurps”).
? The spliceosome is the largest
snRNP,and the exact makeup
differs at different stages of the
splicing reaction
? Three roles of snRNPs in splicing
1,Recognizing the 5’ splice site
and the branch site.
2,Bringing those sites together.
3,Catalyzing (or helping to
catalyze) the RNA cleavage.
RNA-RNA,RNA-protein and
protein-protein interactions are
all important during splicing.
Figure 13-6
RNA-RNA interactions between different
snRNPs,and between snRNPs and pre-mRNA
Topic 3
SPLICING
PATHWAYS
CHAPTER 13 RNA Splicing
Assembly,rearrangement,and
catalysis within the spliceosome,
the splicing pathway (Fig,13-8)
? Assembly step 1
1,U1 recognize 5’ splice site,
2,One subunit of U2AF binds to
Py tract and the other to the 3’
splice site,The former subunits
interacts with BBP and helps it
bind to the branch point.
3,Early (E) complex is formed
Sp
lic
in
g
pa
th
w
ays
? Assembly step 2
1,U2 binds to the branch site,and
then A complex is formed.
2,The base-pairing between the
U2 and the branch site is such
that the branch site A is
extruded(Figure 13-6),This A
residue is available to react
with the 5’ splice site.
Figure 13-8
E complex
A complex
Figure 13-6b
? Assembly step 3
1,U4,U5 and U6 form the tri-snRNP
Particle,
2,With the entry of the tri-snRNP,
the A complex is converted into
the B complex.
Figure 13-8
A complex
B complex
? Assembly step 4
U1 leaves the complex,and U6
replaces it at the 5’ splice site.
U4 is released from the complex,
allowing U6 to interact with U2
(Figure 13-6c).This arrangement
called the C complex.
Figure 13-8Figure 13-6c
B complex
C complex
in which the
catalysis has
not occurred
yet
Catalysis Step 1:
Formation of the C complex
produces the active site,with U2
and U6 RNAs being brought
together
Formation of the active site
juxtaposes the 5’ splice site of
the pre-mRNA and the branch
site,allowing the branched A
residue to attack the 5’ splice
site to accomplish the first
transesterfication reaction.
Catalysis Step 2:
U5 snRNP helps to bring the two
exons together,and aids the
second transesterification
reaction,in which the 3’-OH of
the 5’ exon attacks the 3’ splice
site.
? Final Step:
Release of the mRNA product
and the snRNPs
Figure 13-8
C complex
E complex
A complex
B complex
C complex （没有
该 complex的图）
splicesome-mediated splicing reactions
Figure 13-8
Self-splicing introns reveal that
RNA can catalyze RNA splicing
? Self-splicing introns,the intron
itself folds into a specific
conformation within the
precursor RNA and catalyzes
the chemistry of its own release
and the exon ligation
Sp
lic
in
g
pa
th
w
ays
Adams et al.,Nature 2004,Crystal structure of a
self-splicing group I intron with both exons
Practical definition for self-splicing
introns,the introns that can remove
themselves from pre-RNAs in the
test tube in the absence of any
proteins or other RNAs,
There are two classes of self-
splicing introns,group I and group
II self-splicing introns.
TABLE 13-1 Three class of RNA Splicing
Class Abundance Mechanism Catalytic
Machinery
Nuclear
pre-
mRNA
Very common; used
for most eukaryotic
genes
Two
transesterificat
ion reactions;
branch site A
Major
spliceosom
e
Group
II
introns
Rare; some eu-
Karyotic genes from
organelles and
prokaryotes
Same as pre-
mRNA
RNA
enzyme
encoded
by intron
(ribozyme)
Group I
introns
Rare; nuclear rRNA in
some eukaryotics,
organlle genes,and a
few prokaryotic genes
Two
transesterific-
ation reactions;
exogenous G
Same as
group II
introns
? The chemistry of group II
intron splicing and RNA
intermediates produced are
the same as that of the
nuclear pre-mRNA.
Figure 13-9
Group I introns release a linear
intron rather than a lariat
? Instead of using a branch point A,
group I introns use a free G to attack
the 5’ splice site.
? This G is attached to the 5’ end of the
intron.The 3’-OH group of the 5’ exon
attacks the 5’ splice site.
? The two-step transesterification
reactions are the same as that of
splicing of the group II intron and
pre-mRNA introns.
Sp
lic
in
g
pa
th
w
ays
G instead of A
a linear introna Lariat intron
Figure 13-9
1,Smaller than group II introns
2,Share a conserved secondary
structure,which includes an
“internal guide sequence” base-
pairing with the 5’ splice site
sequence in the upstream exon.
3,The tertiary structure contains a
binding pocket that will
accommodate the guanine
nucleotide or nucleoside cofactor
Group I introns
The similarity of the structures
of group II introns and U2-U6
snRNA complex formed to
process first transesterification
Figure
13-10
How does spliceosome find
the splice sites reliably
Sp
lic
in
g
pa
th
w
ays
Two kinds of splice-site
recognition errors
? Splice sites can be skipped.
?,Pseudo” splice sites could be
mistakenly recognized,
particularly the 3’ splice site,
Figure 13-12
Reasons for the recognition
errors
(1) The average exon is 150 nt,
and the average intron is about
3,000 nt long (some introns are
near 800,000 nt)
? It is quite challenging for the
spliceosome to identify the
exons within a vast ocean of the
intronic sequences,
(2) The splice site consensus
sequence are rather loose,For
example,only AG?G tri-
nucleotides is required for the 3’
splice site,and this consensus
sequence occurs every 64 nt
theoretically,
1,Because the C-terminal tail of
the RNA polymerase II carries
various splicing proteins,co-
transcriptional loading of these
proteins to the newly synthesized
RNA ensures all the splice sites
emerging from RNAP II are
readily recognized,thus
preventing exon skipping,
Two ways to enhance the
accuracy of the splice-site
selection
2,There is a mechanism to
ensure that the splice sites close
to exons are recognized
preferentially,SR proteins bind
to the ESEs (exonic splicing
enhancers) present in the exons
and promote the use of the
nearby splice sites by recruiting
the splicing machinery to those
sites
SR proteins,bound to exonic
splicing enhancers (ESEs),
interact with components of
splicing machinery,recruiting
them to the nearby splice sites,
Figure 13-13
1,Ensure the accuracy and
efficacy of constitutive splicing
2,Regulate alternative splicing
3,There are many varieties of SR
proteins,Some are expressed
preferentially in certain cell
types and control splicing in
cell-type specific patterns
SR proteins are essential for
splicing
Topic 4
ALTERNATIVE
SPLICING
CHAPTER 13 RNA Splicing
? Many genes in higher eukaryotes
encode RNAs that can be spliced
in alternative ways to generate
two or more different mRNAs
and,thus,different protein
products.
Single genes can produce multiple
products by alternative splicing
Al
te
rn
at
ive
sp
lic
in
g
Drosophila DSCAM gene
can be spliced in 38,000
alternative ways
Figure 13-13
Figure 13-15
There are five different ways to
alternatively splice a pre-mRNA
Alternative splicing can be either
constitutive or regulated
? Constitutive alternative splicing,
more than one product is always
made from a pre-mRNA
? Regulative alternative splicing,
different forms of mRNA are
produced at different time,
under different conditions,or in
different cell or tissue types
An example of constitutive
alternative splicing, Splicing of
the SV40 T antigen RNA
Figure 13-16
Alternative splicing is regulated
by activators and repressors
? The regulating sequences,
exonic (or intronic) splicing
enhancers (ESE or ISE) or
silencers (ESS and ISS),The
former enhance and the latter
repress splicing.
? Proteins that regulate splicing
bind to these specific sites for
their action
Al
te
rn
at
ive
sp
lic
in
g
? SR proteins binding to enhancers
act as activators.
(1) One domain is the RNA-
recognition motif (RRM)
(2) The other domain is RS domain
rich in arginine and serine,This
domain mediates interactions
between the SR proteins and
proteins within the splicing
machinery.
? hnRNPs binds RNA and act as
repressors
1,Most silencers are recognized by
hnRNP ( heterogeneous nuclear
ribonucleoprotein) family,
2,These proteins bind RNA,but
lack the RS domains,Therefore,
(1) They cannot recruit the
splicing machinery,(2) they
block the use of the specific
splice sites that they bind.
Regulated alternative splicing
Figure 13-17
Binds at each end
of the exon and
conceals (隐藏 ) it
Coats the RNA and
makes the exons
invisible to the
splicing machinery
An example of repressors,
inhibition of splicing by hnRNPI
Figure 13-18
? The outcome of alternative
splicing:
1,Producing multiple protein
products,called isoforms.
2,Switching on and off the
expression of a given gene,In
this case,one functional
protein is produced by a
splicing pattern,and the non-
functional proteins are resulted
from other splicing patterns,
A small group of intron are
spliced by minor spliceosome
? This spliceosome works on
a minority of exons,and
those have distinct splice-
site sequence,
? The chemical pathway is the
same as the major
spliceosome.
Al
te
rn
at
ive
sp
lic
in
g
Figure 13-19
The AT-AC
spliceosome
U11 and U12 are in
places of U1 and U2,
respectively
Topic 5
EXON
SHUFFLING
CHAPTER 13 RNA Splicing
? All eukaryotes have introns,and
yet these elements are rare in
bacteria,Two likely explanations
for these situation,
1,Introns early model – introns
existed in all organisms but have
been lost from bacteria.
2,Intron late model – introns
never existed in bacteria but
rather arose later in evolution.
Ex
on
sh
uf
fli
ng
Exons are shuffled by recombin-
ation to produce gene encoding
new proteins
? Why have the introns been
retained in eukaryotes?
1,The need to remove introns,
allows for alternative splicing
which can generate multiple
proteins from a single gene,
2,Having the coding sequence of
genes divided into several
exons allows new genes to be
created by reshuffling exon.
? Three observations suggest exon
shuffling actually occur:
1,The borders between exons and
introns within a gene often
coincide with the boundaries
between domains within the
protein encoded by that gene.
For example,DNA-binding protein
Figure 13-21
2,Many genes,and proteins they
encode,have apparently arisen
during evolution in part via exon
duplication and divergence.
3,Related exons are sometimes
found in unrelated genes.
Exons have been reused in genes
encoding different proteins
Figure 13-22
Topic 6
RNA
EDITING
CHAPTER 13 RNA Splicing
RNA editing is another way
of changing the sequence of
an mRNA
I,Site specific deamination,
1,A specifically targeted C
residue within mRNA is
converted into U by the
deaminase.
2,The process occurs only in
certain tissues or cell types
and in a regulated manner.
RN
A
ed
iti
ng
Figure 13-25
The human
apolipoprotein gene
Stop code
In liver In intestines
Figure 13-25
3,Adenosine deamination also
occurs in cells,The enzyme
ADAR (adenosine deaminase
acting on RNA) convert A into
Inosine,Insone can base-pair
with C,and this change can alter
the sequence of the protein,
4,An ion channel expressed in
mammalian brains is the target
of Adenosine deamination.
II Guide RNA-directed uridine
insertion or deletion.
1,This form of RNA editing is
found in the mitochondria of
trypanosomes.
2,Multiple Us are inserted into
specific region of mRNAs after
transcription (or US may be
deleted).
3,The addition of Us to the
message changes codons and
reading frames,completely
altering the,meaning” of the
message.
4,Us are inserted into the message
by guide RNAs (gRNAs),
Having three regions,
anchor– directing the gRNAs to
the region of mRNAs it will edit.
editing region – determining
where the Us will be inserted
poly-U stretch
gRNAs
Figure 13-26
Topic 7
mRNA
TRANSPORT
CHAPTER 13 RNA Splicing
Once processed,mRNA is packaged
and exported from the nucleus into
the cytoplasm for translation
m
RN
A
tr
an
sp
or
t
? All the fully processed mRNAs
are transported to the
cytoplasm for translation into
proteins
? Movement from the nucleus to the
cytoplasm is an active and carefully
regulated process.
? The damaged,misprocessed and
liberated introns are retained in the
nucleus and degraded.
1.A typical mature mRNA carries a
collection of proteins that identifies
it as being ready for transport,
2.Export takes place through the
nuclear pore complex,
3.Once in the cytoplasm,some
proteins are discarded and are
then imported back to the
nucleus for another cycle of
mRNA transport,Some proteins
stay on the mRNA to facilitate
translation.
Figure 13-27
1,Why RNA splicing is important?
2,Chemical reaction,determination of the
splice sites,the products,trans-splicing
3,Spliceosome,splicing pathway and finding
the splice sites
4,Self-splicing introns and mechanisms
5,Alternative splicing and regulation,
alternative spliceosome
6,Two different mechanisms of RNA editing
7,mRNA transport-a link to translation
Key points of the chapter
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