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Chapter13 

RNA splicing



Outline: 

• The chemistry of RNA Splicing 

• The Spliceosome Machinery 

• Splicing Pathways 

• Alternative Splicing 

• Exon Shuffling 

• RNA Editing 

• mRNA Transport



We have tacitly assumed that the coding 

sequence is contiguous:the cordon for one 

amino acid is immediately adjacent to the 

cordon for the next amino acid in the 

polypeptide chain.This is ture in the vast 

majority of cases in bacteria and their phage. 

But it is not always so for eukaryotic 

genes.In those cases,the coding sequence 

is periodically by stretches of noncoding 

sequence. Most of the eukaryotic genes are 

mosaic ( 嵌合体 ), consisting of intervening 

sequences separating the coding sequence



Primary transcript 

Typical eukaryotic gene

Conceptions: 



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.



1.THE CHEMISTRY OF 

RNA SPLICING



1.1 Sequences within the RNA 

Determine Where Splicing Occurs 

The borders between introns and exons are 

marked by specific nucleotide sequences 

within the pre-mRNAs.These sequences 

delineate where splcing will occur. 

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).



Seequences at the intron-exon boundary



1.2 The intron is removed in a Form 

Called a Lariat 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’ 5 splice site. As a 

result, the 5’ 5 exon is released and the 5’-end 5 

of the intron forms a three-way junction 

structure. 

Step 2: The OH of the 5’ 5 exon attacks the 

phosphoryl group at the 3’ 3 splice site. As a 

consequence, the 5’ 5 and 3’ 3 exons are joined 

and the intron is liberated in the shape of a 

lariat.



Step 1 

Step 2



The structure of 

three-way function



1.3 Exons from different RNA 

molecules can be fused by Trans- 

splicing 

• The 5’ 5 splice site of one exon is not always 

joint to the 3’ 3 splice site of the exon.Some 

exons can be skipped,and a given exon is 

joint to one further downstream. 

• Trans-splicing( 

splicing( 反式剪接 ): the process in 

which two exons carried on different RNA 

molecules can be spliced together. It occurs 

in most all the mRNAs of trypanosomes and 

all the mRNA of the nematode worms.



Transsplicing 

Not a lariat but a Y shape branch structure



2 THE SPLICESOME 

MACHINERY

2.1 RNA splicing is carried out 

by a large complex called 

spliceosome 



• The transesterifiction reaction described are 

mediated by the spliceosome which comprises 

about 150 proteins and 5 RNAs. 

• Many functions of the spliceosome are carried 

out by its RNA components. 

• The five RNAs(U1, U2, U4, U5, and U6, 100-300 

nt)in the spliceosome are called small nuclear 

RNAs (snRNAs). 

• The snRNAs is complexed with several 

proteins and these RNA-protein complexes are 

called small nuclear ribonuclear 

proteins(snRNPs).



• The spliceosome is the largest 

complexes made up of these snRNPs, 

but the exact makeup differs at 

different stages of the splicing reaction. 

• Three roles of snRNPs in splicing: 

1. Recognizing the 5’ 5 splice site and the 

branch site. 

2. Bringing those sites together as 

required. 

3. Catalyzing (or helping to catalyze) the 

RNA cleavage and joining reactions.



A.Interaction of U1 

snRNA and the 

5’splice site in the premRNA 

then the splice 

site is recognized by 

the U6 snRNA.. 

B.The snRNP U2 is 

rencognizing the 

branch site. 

C.The RNA:RNA 

pairing between the 

snRNPs U2 and U6. 

D.The same sequence 

within the pre-mRNA is 

recognized by a 

protein at one stage 

and displaced by an 

snRNP at another



3 SPLICING PATHWAYS



3.1 Assembly, , rearrangement, and 

catalysis within the spliceosome: 

the splicing pathway



• Assembly : 

1. U1 recognize 5’ 5 splice site. 

2. One subunit of U2AF binds to Py tract and the other to 

the 3’ 3 splice site. The former subunits interacts with 

BBP and helps it bind to the branch point. 

3. Early (E) complex is formed 

4. U2 binds to the branch site aided by U2AF and 

displacing BBP, and then A complex is formed. 

5. The base-pairing between the U2 and the branch site 

is such that the branch site A is extruded. This A 

residue is available to react with the 5’ 5 splice site. 

6. U4, U5 and U6 form the tri-snRNP Particle. 

7. With the entry of the tri-snRNP, the A complex is 

converted into the B complex. 

8.U1 leaves the complex, and U6 replaces it at the 5’ 5 

splice site.U4 is released from the complex, allowing 

U6 to interact with U2 This arrangement called the C 

complex.


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Catalysis : 

1.Formation of the C complex produces the 

active site, with U2 and U6 RNAs being 

brought together 

2.Formation of the active site juxtaposes the 5’ 5 

splice site of the pre-mRNA and the branch 

site, allowing the branched A residue to attack 

the 5’ 5 splice site to accomplish the first 

transesterfication reaction. 

3.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. 

4.Release of the mRNA product and the snRNPs



3.2 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 there t are two 

classes of self-splicing introns, group I and 

group II self-splicing introns. 

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 RNA 

molecules.

TABLE 13-1 



Three class of RNA Splicing 

Class 

Abundance 

Mechanism 

Catalytic 

Machinery 

Nuclear 

pre- 

mRNA 

Very common; used for 

most eukaryotic genes 

Two 

transesterification 

reactions; branch 

site A 

Major 

spliceosome 

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



• Strictly speaking,self-splicing splicing introns are not 

enzymes(catalysts),because they mediate 

only one round of RNA processing. 

• The chemistry of group II intron splicing and 

the RNA intermediates produced are the 

same as that of the nuclear pre-mRNA.The 

introns uses an A residue within the branch 

site to attack the phosphodiester bond at the 

boundary between its 5’end 5 

and the end of 

the 5’exon5 

exon-that is,at the 5’splice 5 

site.Then it 

forms a lariat and is followed by a second 

reaction in which the newly freed 3’OH 3 

of the 

exon attacks the 3’splice 3 

site,releasing the 

intron as a lariat and fusing the 3’ 3 and 5’ 5 

exons.



3.3Group I introns release a linear 

intron rather than a lariat 

Instead of using a branch point A, group I 

introns use a free G nucleotide or nucleoside. 

This G species is bound by the RNA and its 

3’OH group is present to the 5’ 5 splice site. 

The two-step transesterification reactions are 

the same as that of splicing of the group II 

intron and pre-mRNA introns.



G instead of A 

a Lariat 

intron 

a linear intron



Group I introns 

Group 1 intons are smaller than group II introns and 

share a conserved secondary structure, which 

includes a binding pocket that will accommodate any 

guanine nucleotide or nucleoside as long as it is 

ribose form.In addition to the nucleotide –binding 

pocket,group1 introns contain an “internal guide 

sequence” that base-pairs 

with the 5’ 5 splice site 

sequence thereby determines the precise site at 

which nucleophilic attack by the G nucleotide take 

place. 

The sequence of self-splicing splicing intron is critical for the 

splicing reaction ,and sequence requirment holds 

because the intron must folds into a precise 

structure to perform the reaction chemistry.



The structure of 

the catalytic 

region that 

performs the first 

transesterificatio 

n reaction is verh 

similar in the 

group2 introns 

and the premRNA 

/snRNP 

complex.



3.4 How does spliceosome find the 

splice sites reliably 

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. 

Two kinds of splice-site recognition errors: 

(1) Splice sites can be skipped. 

(2) Other sites could be mistakenly recognized.





Two ways to enhance the accuracy of 

the splice-site selection: 

1. Because the C-terminal C 

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. 

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.



SR proteins are essential for splicing: 

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



4 ALTERNATIVE 

SPLICING



4.1 Single genes can produce multiple 

products by alternative 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.The basic answer is that some 

splice sites are used only some of the 

time,leading to the production of different 

versions of the RNA from different 

transcripts of the same gene.



Alternative splicing in the troponin T gene



Five different ways to 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



Constitutive alternative splicing : 

Splicing of the SV40 T antigen RNA



4.2Alternative 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



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 : 

Most silencers are recognized by hnRNP 

( heterogeneous nuclear 

ribonucleoprotein) family. 

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



Repressors: inhibition of splicing by hnRNPI 

Binds at each end of 

the exon and conceals 

it 

Coats the RNA and 

makes the exons 

invisible to the splicing 

machinery



The outcome of alternative splicing: 

1. Producing multiple protein products from a 

single gene, called isoforms. 

2. Switching on and off the expression of a given 

gene. 

(1)An exon contains a stop codon,when 

incorporated into mRNA,this prematurely 

terminates translation generating a truncated 

polypeptide. 

(2)Alternative splicing used as an on/off switch 

is by regulating the use of an intron,which,when 

retained in the mRNA ,ensure that species is not 

transported out of the nucleus and so is never 

translated.



4.3 A small group of intron are 

spliced by minor spliceosome 

• Some pre-mRNAs are spliced by a low- 

abundance form of spliceosome.This rare 

form contains some components common to 

the major spliceosome but other unique 

components as well. 

• This minor spliceosome recognizes rarely 

occuring introns having consensus 

sequences dinstict from the sequence of 

most pre-mRNA 

• Despite the different splice site and branch 

site sequences recognized by the two 

systems,these major and minor forms of 

spliceosomes both remove introns using the 

same chemical pathway.



U11 and U12 

instead of U1 

and U2, 

The AT- 

AC 

spliceoso 

me 

catalyzed 

splicing



5 EXON SHUFFLING



5.1 Exons are shuffled by 

recombin-ation to produce gene 

encoding new proteins 

• 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.



Why have the introns been retained 

in eukaryotes? 

1.The presence of the introns and the 

need to remove them, 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. 

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 encode protein domains 

The 

dimerzation 

domain and 

DNA-binding 

domain are 

cncoded by 

separate 

exons



Genes made up of parts of other genes 

The same exons are encoded in different proteins



6 RNA EDITING



6.1 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 

deamination.The process occurs only in 

certain tissues or cell types and in a 

regulated manner. 

(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. An ion 

channel expressed in mammalian brains is 

the target of Adenosine deamination.



The deamination of the base cytosine to 

produce uracil



RNA editing by deamination 

Stop code 

In liver 

In intestines



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) .



Three regions of gRNAs: 

anchor– directing the gRNAs to the 

region of mRNAs it will edit. 

editing region – determining where the 

Us will be inserted 

poly-U U stretch



RNA editing by guide RNA mediated U insertion



7 mRNA TRANSPORT



7.1 Once processed, mRNA is packaged 

and exported from the nucleus into the 

cytoplasm for translation 

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.





By 

闻博 

生物科学 200431060189

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