Alternative Splicing

HOW ONE GENE CAN MAKE MANY PROTEINS

Humans have only twice as many genes as the simple fruit fly.

How can this be?

Alternative splicing explains the compact living of our genetic

information as well as the mechanisms behind several human
diseases.
❖ Scientists thought that the complex DNA of a human was made up by
perhaps as many as 150,000 different genes.

❖ The approximation was based on the number of different transcripts,

mRNAs, which had been found in humans, assuming that there should be
one gene for each mRNA.

❖ When all the sequencing was done, the result was merely 32,000 human
genes.
❖ In comparison, the fruit fly Drosophila has 14,000 genes and the
nematode Caenorhabditis elegans has 19,000.

❖ The number of human expressed-sequence (mRNA) forms was much

higher than the number of genes.
❖ How can the much greater size and complexity of humans be encoded
in only twice the number of genes required by a fly?

❖ One way to explain this paradox is to point out that the number of possible
proteins from the genome can far exceed the number of genes if a large
percentage of the genes have the ability to encode multiple proteins.

❖ This expansion of the proteome can be accomplished through

alternative precursor messenger RNA (pre mRNA) splicing.
Alternative splicing is a process which allows the production of a variety
of different proteins from one gene only.
 Nuclear Splice Sites Are Short Sequences

 There exist minor introns relative to the major introns that follow
the GU-AG rule.

 Minor introns follow a general AU-AC rule with a different set of

consensus sequences at the exon–intron boundaries.
 Splice Sites Are Read in Pairs

Figure 21.04: Splicing junctions are recognized only in the correct pairwise combinations.
 Pre-mRNA Splicing Proceeds Through a Lariat

 Splicing requires the 5′ and 3′ splice sites and a branch site just upstream of
the 3′ splice site.

 The branch sequence is conserved in yeast but less well conserved in

multicellular eukaryotes.

 A lariat is formed when the intron is cleaved at the 5′ splice site, and the 5′ end
is joined to a 2′ position at an A at the branch site in the intron.
Pre-mRNA Splicing Proceeds Through a Lariat

The intron is released as a lariat when

it is cleaved at the 3′ splice site, and
the left and right exons are then
ligated together.

Figure : Splicing occurs in two stages. First the

5’ exon is cleaved off, and then it is joined to
the 3’ exon.
snRNAs Are Required for Splicing

▪ small nuclear RNA (snRNA; snurps) – One of many

small RNA species confined to the nucleus; several
of them are involved in splicing or other RNA
Figure: The
processing reactions.
spliceosome is
~12 MDa. Five
snRNPs ▪ The five snRNPs (small nuclear ribonucleoproteins)
account for involved in splicing are U1, U2, U5, U4, and U6.
almost half of
the mass. ▪ Together with some additional proteins, the snRNPs
form the spliceosome.
 Alternative Splicing Is a Rule, Rather Than an Exception, in Multicellular
Eukaryotes

▪ Specific exons or exonic sequences may be excluded or included in the

mRNA products by using alternative splicing sites.

▪ Alternative splicing contributes to structural and functional diversity of gene

products.
Alternative Splicing Is a Rule, Rather Than an Exception, in Multicellular Eukaryotes

Figure : Different modes of alternative splicing.

Exon Skipping: This mechanism is also known as cassette exon,
where an exon is spliced out of the gene during the
transcription. An example would be the dsx gene in D.
melanogaster (fruit fly).

Mutually Exclusive Exons: In this case only one of two

consecutive exons is retained during transcription. An
example is the regulation of exons 8a and 8 in the CaV1.2
calcium channels.

In Timothy syndrome, the alternate forms of these two exons

can lead to different symptoms of the disease, which causes
disruption of the calcium homeostasis needed for muscle
contraction. However, both exons cannot exist in patients;
only one of them is transcribed, though both are present in
the gene.
Alternative 3’ Acceptor Sites: The splice junction at the 3’
end is used, changing the 5’ boundary of the
downstream exon.
An example is the Transformer (Tra) activator protein
present in females of D. melanogaster (fruit fly).

Alternative 5’ Donor Sites: The splice junction at the 5’ is used,

changing the 3’ boundary of the upstream exon. While
alternative acceptor sites lead to small variations in protein
sequences, alternative donor sites can lead to drastic
differences in protein sequence and structure because it can
cause frameshifts.

An example would be the alternative donor site splicing of

BTNL2 gene. The use of the upstream site, instead of the
downstream site, leads to an abridged protein without the C-
terminal IgC domain or the transmembrane helix. This results in
predisposition to chronic inflammatory disease.
Intron Retention: Similar to exon skipping, the exon is
retained in the mRNA, but unlike exon skipping, the exon
is not flanked by introns.

If introns did exist, they are often encoded in the coding

regions among the amino acids of close by exons, the
stop codon, or a shift in the reading frame causing the
protein to become non-functional. This is the least
common mechanism of alternative splicing.
Sex determination in Drosophila involves a series of alternative splicing events in
genes encoding successive products of a pathway.

1) Male include exon-3 of sxl gene which have termination

codon; Therefore, ultimately no SXL protein is produced.
2) Female exclude exon-3 of sxl gene
3) SXL protein in female
4) SXL protein change the splicing of Tra gene
5) Tra produce Tra2 gene
6) Tra + Tra 2 produce dsx (double sex) gene. Tra + Tra 2
interacts with a splicing enhancer in the fourth exon of
the double-sex pre-mRNA and activates use of the
upstream 3′ splice site.
7) DSX protein suppress male genes & promote female
development

Sex determination in D. melanogaster involves a pathway in

which different splicing events occur in females. Blocks at
any stage of the pathway result in male development.

Figure : Sex determination in D. melanogaster involves a pathway in which different

splicing events occur in females.
Cascade of alternative splicing events in the sex
determination pathway of Drosophila. For
simplicity, only those exons and introns involved in
alternative splicing events are shown. In female
flies, the sex lethal protein regulates its own
synthesis and that of the tra protein by blocking
the use of the 3′ splice site of the third and
second exons of the sex-lethal and tra pre-
mRNAs, respectively. Both of these exons contain
a stop codon, as indicated.

The tra protein, together with tra-2, interacts with

a splicing enhancer in the fourth exon of the
double-sex pre-mRNA and activates use of the
upstream 3′ splice site. Exons are depicted as
numbered boxes, and introns are depicted as
solid lines. Dashed lines above the introns
represent splicing events in females, and those
below indicate splicing events in males. The sex-
lethal (sxl), transformer (tra), and tra-2 proteins are
indicated by circles. Polyadenylation is indicated
by p(A).
Splicing Can Be Regulated by Exonic and Intronic Splicing
Enhancers and Silencers

 Alternative splicing is often associated with weak splice sites.

 Sequences surrounding alternative exons are often more

evolutionarily conserved than sequences flanking constitutive
exons.

 Specific exonic and intronic sequences can enhance or suppress

splice site selection.
Figure : RNA is modified in the nucleus by additions to the 5’ and 3’ ends and by splicing to remove the
introns.
 heterogeneous nuclear RNA (hnRNA) – RNA that comprises
transcripts of nuclear genes made by RNA polymerase II; it has a
wide size distribution and low stability.

 hnRNP – The ribonucleoprotein form of hnRNA (heterogeneous

nuclear RNA), in which the hnRNA is complexed with proteins.
 Pre-mRNAs are not exported until processing is complete; thus they are
found only in the nucleus.
 The 5′ End of Eukaryotic mRNA Is Capped

 A 5′ cap is formed by adding a G to the terminal base of the transcript via a 5′–
5′ link.
 The capping process takes place during transcription and may be important for
release from pausing of transcription.

Figure : The cap blocks the 5’

end of mRNA and is
methylated at several
positions.
 The 5′ cap of most mRNA is monomethylated, but some small
noncoding RNAs are trimethylated.

 The cap structure is recognized by protein factors to influence

mRNA stability, splicing, export, and translation.
  A surprisingly common process !!!

 Alternative splicing has long been regarded as a rather rare event in

eukaryotic genomes. For example, it was estimated to occur in less than
5% of human genes.

 However, recent analyses of vast amounts of transcript data in human

and other organisms suggest that alternative splicing is widespread in
mammalian genomes. In humans, it is estimated that alternative splicing
occurs in more than 60% of genes.

 The one gene, one protein central dogma, a governing theory of

modern molecular biology which has profound impact on biologists’
thinking, therefore has to be revisited.

 As a result, our view towards many biological processes, such as protein

interaction and gene expression, has to be adjusted.
 Alternativesplicing is a powerful means of enhancing
protein diversity. It is estimated that over 70% of all
genes are alternatively spliced as a means for
producing functionally diverse proteins from a single
gene.

 The majority of metazoan genes encode

pre-mRNAs that are alternatively spliced to produce
anywhere from two to tens of thousands of mRNA
isoforms.
 One of the most dramatic examples of alternative splicing is
the Dscam (Down syndrome cell adhesion molecule ) gene
in Drosophilia.

 This single gene contains some 116 exons of which 17 are

retained in the final mRNA.

 Some exons are always included;

 Others are selected from an array. Theoretically this system
is able to produce 38,016 different proteins. And, in fact,
over 18,000 different ones have been found in Drosophila.
 Alternative splicing plays an important role in regulating
gene expression.

 In the last decade it has been shown that alternative splicing

determines ------------
 binding properties,
 intracellular localization,
 enzymatic activity,
 protein stability and
 posttranslational modifications of a large number of proteins.
 Many proteins are comprised of several domains, or
modules, that serve a particular function.

 For example, one domain may help the protein bind to

another protein, while another domain gives the protein
enzymatic activity.

 In the genome, domains correspond to exons.

 By alternative splicing exons, i.e. protein domains, can be

mixed and matched, altering the nature of the protein.
 The consequences of alternative splicing fall into two major
categories:
 Protein-level alterations and
 Transcript-level modifications.

 On the protein level, alternative splicing generates splice

variants that give rise to different protein products.

 For example, a shortened protein product due to a frame

shift introduced by an alternate exon or a protein product
with a different functional domain due to the inclusion of a
specific exon from a mutually exclusive group of exons.
 On the transcript level, alternative splicing produces splice
variants that have different translation or stability profiles.

 For example, a transcript with a longer lifespan would

prolong the availability of the corresponding protein.

 Thus, it plays an extremely important role in expanding

protein diversity and might therefore partially explain the
apparent discrepancy between gene number and organism
complexity.
 Alternative splicing and human disease

 Alternative splicing events have been implicated in a large number

of human pathologies, including
neurodegenerative,
cardiovascular,
respiratory and
metabolic diseases, as well as
cancer and
Alzheimer’s disease,

 and are promising targets for therapeutic intervention.

 In tissues with complex activities, like the brain, there is a
demand for a large number of proteins to perform different
functions.

 Any defect in alternative splicing can cause severe

problems for the cell since the protein composition is
altered, and many neurological diseases are actually
caused by defects in alternative splicing.

 These defects can be divided into two major groups:

Primary and secondary splicing defects
 Primary splicing defects

 In primary splicing defects, sequences on the pre-mRNA

which are important for splicing are mutated.
Consequently, correct splicing cannot occur.
this fig
 Many point mutations causing human disease are considered to cause
defects in pre-mRNA splicing.

 Examples are ataxia-telangiectasia (Ataxia telangiectasia (A-T) is an autosomal

recessive disorder primarily characterized by cerebellar degeneration, telangiectasia,
immunodeficiency, cancer susceptibility and radiation sensitivity) and neurofibromatosis.
(a genetic disorder that causes tumors to form on nerve tissue. These tumors can develop
anywhere in your nervous system, including your brain, spinal cord and nerves.)

 Half of the patients suffering from those diseases carry mutations that
effect pre-mRNA splicing.

 Another example for primary splicing disorders is found in fronto-temporal

dementia (uncommon brain disorders that primarily affect the frontal and temporal lobes of the
brain. These areas of the brain are generally associated with personality, behavior and language.
Portions of these lobes shrink (atrophy) patients. This syndrome is an autosomal
dominant disorder linked to chromosome.
 Secondary splicing defects

 In
secondary splicing disorders, a regulatory factor
which is essential for the process of splicing, is
mutated and disturbs splicing activity.

 Differences in the regulation of splicing activator

and/or repressor proteins can have an effect on the
alternative splicing pathway.
 Secondary splicing defects
this fig

In this pre-mRNA two different splice sites (AG)

can be used for splicing. The two resulting
products after splicing and translation (Protein Z
and Protein Y) are different depending on the
splice site which was used.

In the normal situation (panel A) one splice site is

partly blocked by a protein factor X. Only the
second site at the end of the intron is easily
accessible and therefore one of the proteins,
Protein Z, is produced in larger amounts than
Protein Y.

After a mutation (panel B), the splicing regulator X

cannot bind to the pre-mRNA any more. Both AG
splice sites are accessible and can be used for
splicing. When the percentage of Protein Z is
reduced and Protein Y levels increase, this causes
disease.
One example for a secondary splicing defect is the Prader Willi Syndrome (PWS), a
genetic disorder characterized by intellectual and behavioural disturbances.

The disease may arise from the absence of a small RNA molecule which normally
regulates pre-mRNA splicing of the serotonin receptor RNA.

The enzyme acetylcholine esterase (AChE) is a combinatorial series of proteins which is

produced via alternative splicing events.

Three alternatively spliced isoforms have been identified which are involved in the
progression of neurodegenerative disorders like Alzheimer’s disease and Parkinson’s
disease.