Introduction What RNA Splicing??? Discovery Types Alternative Splicing Mechanism Regulatory element And protein Splicing repression Splicing activation Significance Diseases Conclusion Refrences

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ALTERNATIVE SPLICING
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

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Synopsis
Alternative Splicing
Introduction
What RNA Splicing???
Discovery
Types
Alternative Splicing
Mechanism
Regulatory element And protein
Splicing repression
Splicing activation
Significance
Diseases
Conclusion
Refrences

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Introduction-
Alternative Splicing
Alternative splicing (or differential splicing) is a
process by which the exons of the RNA produced by
transcription of a gene a primary gene transcript or
pre-mRNA are reconnected in multiple ways during
RNA splicing.
The resulting different mRNAs may be translated into
different protein isoforms; thus, a single gene may
code for multiple proteins.

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What is RNA splicing?

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Genetic information is transferred from genes to the proteins
they encode via a “messenger” RNA intermediate
DNA GENE
messenger RNA
(mRNA)
protein
transcription
translation

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Most genes have their protein-coding information
interrupted by non-coding sequences called “introns”.
The coding sequences are then called “exons”
DNA GE NE
intron
exon 1 exon 2
transcription
precursor-mRNA
(pre-mRNA)
intron

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The intron is also present in the RNA copy of
the gene and must be removed by a process
called “RNA splicing”
protein
translation
mRNA
RNA splicing
pre-mRNA
intron

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Discovery
Alternative splicing was first observed in 1977. Adenoviruses
produce two different primary transcripts, one early in the life
cycle and one later, after DNA replication. Researchers found
that the primary RNA transcript produced by adenovirus type 2
in the late phase was spliced in different ways, resulting in
mRNAs encoding different viral proteins.
In 1981, the first example of alternative splicing in a
transcript from a normal, endogenous gene was characterized.
The gene encoding the thyroid hormone calcitonin was
found to be alternatively spliced in mammalian cells. Examples
of alternative splicing in immunoglobin gene transcripts in
mammals were also observed in the early 1980s.
Since then, alternative splicing has been found to be
ubiquitous in eukaryotes.

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Types of
splicing

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Alternative Splicing
5’ UTR 3’ UTRCoding Sequence
mRNA 1 mRNA 2
Isoform 1 Isoform 2
Transcription
Translation
Folding
AS region

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Alternative splicing
mechanisms
General splicing mechanism•When the pre-mRNA has been transcribed from the DNA.The
exons to be retained in the mRNA are determined during the
splicing process. The regulation and selection of splice sites are
done by trans-acting splicing activator and splicing repressor
proteins.
•The typical eukaryotic nuclear intron has consensus sequences
defining important regions. Each intron has GU at its 5' end. Near
the 3' end there is a branch site. The nucleotide at the branch
point is always an A; the consensus around this sequence varies
somewhat.
•The branch site is followed by a series of pyrimidines, or
polypyrimidine tract, then by AG at 3' end.
•Splicing of mRNA is performed by an RNA and protein complex
known as the spliceosome, containing snRNPs designated U1, U2,
U4, U5, and.
•U1 binds to 5' GU and U2 binds to branch site (A) with the
assistance of the U2AF protein factors. The complex at this stage
is known as the spliceosome A complex. Formation of the A
complex is usually the key step in determining the ends of the
intron to be spliced out, and defining the ends of the exon to be
retained.

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Regulatory elements and proteins
Splicing repression

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Splicing activation

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Examples
Exon skipping: Drosophila dsx

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Alternative acceptor sites:
Drosophila Transformer
•Pre-mRNAs of the Transformer (Tra) gene of
Drosophila melanogaster undergo alternative
splicing via the alternative acceptor site mode.
•The gene Tra encodes a protein that is
expressed only in females. The primary transcript
of this gene contains an intron with two possible
acceptor sites. In males, the upstream acceptor
site is used.
•This causes a longer version of exon 2 to be
included in the processed transcript, including an
early stop codon. The resulting mRNA encodes a
truncated protein product that is inactive.
Females produce the master sex determination
protein Sex lethal (Sxl).

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Exon definition: Fas recepto
Alternative splicing of the Fas receptor pre-mRNAMultiple
isoforms of the Fas receptor protein are produced by alternative
splicing.
An mRNA including exon 6 encodes the membrane-bound form
of the Fas receptor, which promotes apoptosis, or programmed
cell death. Increased expression of Fas receptor in skin cells
chronically exposed to the sun, and absence of expression in
skin cancer cells, suggests that this mechanism may be
important in elimination of pre-cancerous cells in humans.
If exon 6 is skipped, the resulting mRNA encodes a soluble Fas
protein that does not promote apoptosis. The inclusion or
skipping of the exon depends on two antagonistic proteins, TIA-
1 and polypyrimidine tract-binding protein (PTB).
•Binding of TIA-1 protein to an intronic splicing enhancer site
stabilizes binding of the U1 snRNP. The resulting 5' donor site
complex assists in binding of the splicing factor U2AF to the 3'
splice site upstream of the exon, through a mechanism that is
not yet known Exon 6 contains a pyrimidine-rich exonic splicing
silencer, ure6, where PTB can bind. If PTB binds, it inhibits the
effect of the 5' donor complex on the binding of U2AF to the
acceptor site, resulting in exon skipping .

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Repressor-activator competition: HIV-1 tat exon

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Significance
•Alternative splicing is one of several exceptions to the original idea that one DNA
sequence codes for one polypeptide .
• It might be more correct now to say "One gene – many polypeptides.” External
information is needed in order to decide which polypeptide is produced, given a DNA
sequence and pre-mRNA..
• It has been proposed that for eukaryotes alternative splicing was a very important step
towards higher efficiency, because information can be stored much more economically.
•It also provides evolutionary flexibility.
•A single point mutation may cause a given exon to be occasionally excluded or included
from a transcript during splicing, allowing production of a new protein isoform without
loss of the original protein
• Research based on the Human Genome Project and other genome sequencing has
shown that humans have only about 30% more genes than the roundworm
Caenorhabditis elegans, and only about twice as many as the fly Drosophila melanogaster.
•This finding led to speculation that the perceived greater complexity of humans, or
vertebrates generally, might be due to higher rates of alternative splicing in humans than
are found in invertebrates.
•However, a study on samples of 100,000 ESTs each from human, mouse, rat, cow, fly (D.
melanogaster), worm (C. elegans), and the plant Arabidopsis thaliana found no large
differences in frequency of alternatively spliced genes among humans and any of the
other animals tested.

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Alternative splicing and disease
Changes in the RNA processing machinery may lead to mis-splicing of multiple
transcripts, while single-nucleotide alterations in splice sites or cis-acting splicing
regulatory sites may lead to differences in splicing of a single gene, and thus in the
mRNA produced from a mutant gene's transcripts. A probabilistic analysis indicates that
over 60% of human disease-causing mutations affect splicing rather than directly
affecting coding sequences.
Abnormally spliced mRNAs are also found in a high proportion of cancerous cells. Until
recently, it was unclear whether such aberrant patterns of splicing played a role in
causing cancerous growth, or were merely a consequence of cellular abnormalities
associated with cancer.
It has been shown that there is actually a reduction of alternative splicing in
cancerous cells compared to normal ones, and the types of splicing differ; for instance,
cancerous cells show higher levels of intron retention than normal cells, but lower levels
of exon skipping. Some of the differences in splicing in cancerous cells may result from
changes in phosphorylation of trans-acting splicing factors.
One study found that a relatively small percentage of alternative splicing variants were
significantly higher in frequency in tumor cells than normal cells, suggesting that there
is a limited set of genes which, when mis-spliced, contribute to tumor development
One example of a specific splicing variant associated with cancers is in one of the
human DNMT genes. Recent provocative studies point to a key function of chromatin
structure and histone modifications in alternative splicing regulation

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CYSTIC FIBROSIS

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References
Cell and molecular biology by Gerald Karp
Molecular biology of the gene by J.d. Watson
Gene VIII by Benjamine Lewine
Internet source
www.alternativesplicingwiki.com
www.sparknotes.com
www.googleimages.com