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
junction
Figure 13-4
This figure has an error
Intron
5’ end
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 (并置 ) 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 (转酯 ) 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
1st reaction
2nd reaction
E complex
A complex
B complex
C complex （没有该 complex的图）
splicesome-mediated splicing reactions
Figure 13-8
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
(组成性剪接 ).
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
Different ways of alternative splicing
Figure 13-14
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
Al
te
rn
at
ive
sp
lic
in
g
The regulating sequences,
exonic (or intronic) splicing
enhancers (ESE or ISE) or
silencers (ESS and ISS),
Activators are proteins bind to
enhancers to enhance splicing,
Repressors are proteins bind to
silencers to repress splicing.
SR proteins are splicing activators
and contain two domains.
(1) One domain is the RNA-recognition
motif (RRM),which is responsible for
RNA binding,
(2) The other domain is the RS domain
[rich in arginine and serine],which
mediates interactions between the SR
proteins and proteins within the
splicing machinery to promote splicing
at the nearby splice sites.
hnRNPs are splicing 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
Two models for the action of a repressor
hnRNPI/PTB in inhibiting splicing
Figure 13-18
The outcome of alternative splicing
(可变剪接的结果 /生物学功能 )
1,Producing multiple protein products,
called isoforms,They can have similar,
distinct or antagonistic functions,
[One gene encodes multiple functions]
2,Switching on and off the expression of
a given gene that encodes only one
function,[When the exon containing a
stop is included to produce
nonfunctional protein,or the intron is
included to prevent mRNA transport]
A small group of intron are
spliced by minor spliceosome
It splices introns harboring determinant
sequences distinct from those recognized
by the major spliceosome,
It is known AT-AC spliceosome,The
termini of the originally identified
introns that is splice contain AU at 5’ss,
and AC at the 3’ ss,
The chemical pathway is the same as
the major spliceosome,but U11 and U12
are used in places of U1 and U2,
respectively.
Al
te
rn
at
ive
sp
lic
in
g
Figure 13-19
The AT-AC
spliceosome
Figure 13-20 Sequences conserved
in different kinds of introns.
Trans-splicing
Topic 5,Self-splicing
introns
自剪接内含子
CHAPTER 13 RNA Splicing
Self-splicing introns reveal that
RNA can catalyze splicing
Self-splicing introns,
---Introns that can fold into a
specific conformation within the
precursor RNA,and catalyze the
chemistry of their own release and
the exon ligation.
---They can remove themselves
from pre-RNAs in the absence of
any proteins or other RNAs in vitro,
Sp
lic
in
g
pa
th
w
ays
fold into a specific
conformation
catalyze the chemical
reaction using metal
ions as cofactors
There are two classes of self-
splicing introns:
group I self-splicing introns
group II self-splicing introns.
TABLE 13-1 Three classes of RNA Splicing
Class Abundance Mechanism Catalytic
Machinery
Nuclear
pre-
mRNA
Very common; most
eukaryotic genes
Two sequential
transesterification
reactions; branch
site A
spliceosome
Group II
introns
Rare; some eukaryotic
genes from organelles
and prokaryotes
Same as pre-mRNA RNA enzyme
encoded by
intron
(ribozyme)
Group I
introns
Rare; nuclear rRNA in
some eukaryotes,
organelle genes,and a
few prokaryotic genes
Two sequential
transesterification
reactions;
exogenous G
Same as group
II introns
Figure 13-9
The chemistry of group II intron splicing and
RNA intermediates produced are the same as
that of the nuclear pre-mRNA.
The similarity of the structures of group
II introns and U2-U6 snRNA complex
formed to process first transesterification
Figure 13-10
Group I introns release a linear
intron rather than a lariat
During the 1st transesterification reaction,
group I introns use a free G,instead of
using a branch point A,to attack the 5’
splice site.
As a result,this G is attached to the 5’
end of the intron.
A 3’-OH group is resulted at the 5’ exon,
which then attacks the 5’ splice site for
the 2nd transesterification reaction,This
is the same as that of splicing of the
group II 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,Their tertiary structure contains a
binding pocket that will accommodate
the guanine nucleotide or nucleoside
cofactor.
Group I introns
Adams et al.,Nature 2004,Crystal structure of a self-
splicing group I intron with both exons.
Group I intron website,www.rna.whu.edu.cn/gissd
2D structure 3D structure
Topic 6
RNA
EDITING
CHAPTER 13 RNA Splicing
RNA editing is another way
of changing the sequence of
an mRNA at the RNA level
I,Site specific deamination (位点特异性去氨反应 ):
1,A specifically targeted C residue
within mRNA is converted into U
by the deaminase (脱氨酶 ),The
process occurs only in certain
tissues or cell types and in a
regulated manner.
RN
A
ed
iti
ng
Figure 13-25
Stop code
In liver In intestines
Figure 13-25 RNA editing by deamination,
The human apolipoprotein gene
2,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,
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 mRNA 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 RNA editing
by gRNA-mediated U
insertion
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 including SR
protein 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
Maniatis and Reed,Nature 2002,416:499-506
补充材料
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
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
junction
Figure 13-4
This figure has an error
Intron
5’ end
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 (并置 ) 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 (转酯 ) 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
1st reaction
2nd reaction
E complex
A complex
B complex
C complex （没有该 complex的图）
splicesome-mediated splicing reactions
Figure 13-8
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
(组成性剪接 ).
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
Different ways of alternative splicing
Figure 13-14
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
Al
te
rn
at
ive
sp
lic
in
g
The regulating sequences,
exonic (or intronic) splicing
enhancers (ESE or ISE) or
silencers (ESS and ISS),
Activators are proteins bind to
enhancers to enhance splicing,
Repressors are proteins bind to
silencers to repress splicing.
SR proteins are splicing activators
and contain two domains.
(1) One domain is the RNA-recognition
motif (RRM),which is responsible for
RNA binding,
(2) The other domain is the RS domain
[rich in arginine and serine],which
mediates interactions between the SR
proteins and proteins within the
splicing machinery to promote splicing
at the nearby splice sites.
hnRNPs are splicing 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
Two models for the action of a repressor
hnRNPI/PTB in inhibiting splicing
Figure 13-18
The outcome of alternative splicing
(可变剪接的结果 /生物学功能 )
1,Producing multiple protein products,
called isoforms,They can have similar,
distinct or antagonistic functions,
[One gene encodes multiple functions]
2,Switching on and off the expression of
a given gene that encodes only one
function,[When the exon containing a
stop is included to produce
nonfunctional protein,or the intron is
included to prevent mRNA transport]
A small group of intron are
spliced by minor spliceosome
It splices introns harboring determinant
sequences distinct from those recognized
by the major spliceosome,
It is known AT-AC spliceosome,The
termini of the originally identified
introns that is splice contain AU at 5’ss,
and AC at the 3’ ss,
The chemical pathway is the same as
the major spliceosome,but U11 and U12
are used in places of U1 and U2,
respectively.
Al
te
rn
at
ive
sp
lic
in
g
Figure 13-19
The AT-AC
spliceosome
Figure 13-20 Sequences conserved
in different kinds of introns.
Trans-splicing
Topic 5,Self-splicing
introns
自剪接内含子
CHAPTER 13 RNA Splicing
Self-splicing introns reveal that
RNA can catalyze splicing
Self-splicing introns,
---Introns that can fold into a
specific conformation within the
precursor RNA,and catalyze the
chemistry of their own release and
the exon ligation.
---They can remove themselves
from pre-RNAs in the absence of
any proteins or other RNAs in vitro,
Sp
lic
in
g
pa
th
w
ays
fold into a specific
conformation
catalyze the chemical
reaction using metal
ions as cofactors
There are two classes of self-
splicing introns:
group I self-splicing introns
group II self-splicing introns.
TABLE 13-1 Three classes of RNA Splicing
Class Abundance Mechanism Catalytic
Machinery
Nuclear
pre-
mRNA
Very common; most
eukaryotic genes
Two sequential
transesterification
reactions; branch
site A
spliceosome
Group II
introns
Rare; some eukaryotic
genes from organelles
and prokaryotes
Same as pre-mRNA RNA enzyme
encoded by
intron
(ribozyme)
Group I
introns
Rare; nuclear rRNA in
some eukaryotes,
organelle genes,and a
few prokaryotic genes
Two sequential
transesterification
reactions;
exogenous G
Same as group
II introns
Figure 13-9
The chemistry of group II intron splicing and
RNA intermediates produced are the same as
that of the nuclear pre-mRNA.
The similarity of the structures of group
II introns and U2-U6 snRNA complex
formed to process first transesterification
Figure 13-10
Group I introns release a linear
intron rather than a lariat
During the 1st transesterification reaction,
group I introns use a free G,instead of
using a branch point A,to attack the 5’
splice site.
As a result,this G is attached to the 5’
end of the intron.
A 3’-OH group is resulted at the 5’ exon,
which then attacks the 5’ splice site for
the 2nd transesterification reaction,This
is the same as that of splicing of the
group II 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,Their tertiary structure contains a
binding pocket that will accommodate
the guanine nucleotide or nucleoside
cofactor.
Group I introns
Adams et al.,Nature 2004,Crystal structure of a self-
splicing group I intron with both exons.
Group I intron website,www.rna.whu.edu.cn/gissd
2D structure 3D structure
Topic 6
RNA
EDITING
CHAPTER 13 RNA Splicing
RNA editing is another way
of changing the sequence of
an mRNA at the RNA level
I,Site specific deamination (位点特异性去氨反应 ):
1,A specifically targeted C residue
within mRNA is converted into U
by the deaminase (脱氨酶 ),The
process occurs only in certain
tissues or cell types and in a
regulated manner.
RN
A
ed
iti
ng
Figure 13-25
Stop code
In liver In intestines
Figure 13-25 RNA editing by deamination,
The human apolipoprotein gene
2,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,
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 mRNA 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 RNA editing
by gRNA-mediated U
insertion
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 including SR
protein 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
Maniatis and Reed,Nature 2002,416:499-506
补充材料
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
