COMPILED AND CIRCULATED BY DR.

POULAMI ADHIKARY MUKHERJEE, ASSISTANT

PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

Split genes and splicing

mechanism
BY
DR. POULAMI ADHIKARY MUKHERJEE
ASSISTANT PROFESSOR
DEPARTMENT OF ZOOLOGY
NARAJOLE RAJ COLLEGE
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PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

A split gene (also called an interrupted gene) is a gene that

contains expressed regions of DNA called exons, split with
unexpressed regions called introns (also called intervening
regions).

Exons provide instructions for coding proteins, which

create mRNA necessary for the synthesis of proteins.
Introns are removed by recognition of the donor site (5'
end) and the splice acceptor site (3' end).
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The architecture of the interrupted gene allows for the

process of alternative splicing, where various mRNA
products can be produced from a single gene.

The function of introns are still not fully understood and

are called noncoding or junk DNA.

Non-coding DNA sequences are components of an

organism's DNA that do not encode protein sequences.
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Some non-coding DNA is transcribed into functional non-

coding RNA molecules (e.g. transfer RNA, ribosomal RNA,
and regulatory RNAs).

Other functions of non-coding DNA include the

transcriptional and translational regulation of protein-
coding sequences, scaffold attachment regions, origins of
DNA replication, centromeres and telomeres. Its RNA
counterpart is non-coding RNA.
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The amount of non-coding DNA varies greatly among

species. Often, only a small percentage of the genome is
responsible for coding proteins, but an increasing
percentage is being shown to have regulatory functions.
So, this non-functional portion has been called "junk DNA".

An exon is any part of a gene that will encode a part of the

final mature RNA produced by that gene after introns have
been removed by RNA splicing.
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The term exon refers to both the DNA sequence within a

gene and to the corresponding sequence in RNA
transcripts.

In RNA splicing, introns are removed and exons are

covalently joined to one another as part of generating the
mature messenger RNA. Just as the entire set of genes for a
species constitutes the genome, the entire set of exons
constitutes the exome.
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An intron (for intragenic region) is any nucleotide

sequence within a gene that is removed by RNA
splicing during maturation of the final RNA product.

In other words, introns are non-coding regions of an RNA

transcript, or the DNA encoding it, that are eliminated by
splicing before translation.

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The word intron is derived from the term intragenic

region, i.e. a region inside a gene. The term intron refers to
both the DNA sequence within a gene and the
corresponding sequence in RNA transcripts. Sequences
that are joined together in the final mature RNA after RNA
splicing are exons.

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Introns are found in the genes of most organisms and

many viruses and can be located in a wide range of genes,
including those that generate proteins, ribosomal
RNA (rRNA) and transfer RNA (tRNA). When proteins are
generated from intron-containing genes, RNA splicing
takes place as part of the RNA processing pathway that
follows transcription and precedes translation.

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Gene Splicing:

Gene splicing is a post-transcriptional modification in

which a single gene can code for multiple proteins. Gene
Splicing is done in eukaryotes, prior to mRNA translation,
by the differential inclusion or exclusion of regions of
pre-mRNA. Gene splicing is an important source of
protein diversity. During a typical gene splicing event,
the pre-mRNA transcribed from one gene can lead to

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different mature mRNA molecules that generate multiple

functional proteins. Thus, gene splicing enables a single
gene to increase its coding capacity, allowing the
synthesis of protein isoforms that are structurally and
functionally distinct. Gene splicing is observed in high
proportion of genes. In human cells, about 40-60% of the
genes are known to exhibit alternative splicing.

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Gene Splicing Mechanism:

There are several types of common gene splicing events.

The events are carried out in a series of reactions which
are catalyzed by the spliceosome, a complex of small
nuclear ribonucleoproteins (snRNPs). Self-splicing
introns, or ribozymes capable of catalyzing their own
excision from their parent RNA molecule, also exist.
These are the events that can simultaneously occur in the

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genes after the mRNA is formed from the transcription

step of the central dogma of molecular biology.

Exon Skipping: This is the most common known gene

splicing mechanism in which exon(s) are included or
excluded from the final gene transcript leading to
extended or shortened mRNA variants. The exons are the
coding regions of a gene and are responsible for

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producing proteins that are utilized in various cell types

for a number of functions.

Intron Retention: An event in which an intron is

retained in the final transcript. In humans 2-5 % of the
genes have been reported to retain introns. The gene
splicing mechanism retains the non-coding (junk)
portions of the gene and leads to a demornity in the
protein structure and functionality.
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Alternative 3' Splice Site and 5' Splice Site: Alternative

gene splicing includes joining of different 5' and 3' splice
site. In this kind of gene splicing, two or more alternative
5' splice site compete for joining to two or more
alternate 3' splice site.

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Splicing pathways:
Several methods of RNA splicing occur in nature; the type of
splicing depends on the structure of the spliced intron and the
catalysts required for splicing to occur.

Self-splicing mechanism: (Introns mediated splicing)

Self-splicing occurs for rare introns that form a ribozyme,
performing the functions of the spliceosome by RNA alone.
There are three kinds of self-splicing introns, Group I, Group
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II and Group III. Group I and II introns perform splicing similar

to the spliceosome without requiring any protein. This
similarity suggests that Group I and II introns may be
evolutionarily related to the spliceosome. Self-splicing may
also be very ancient, and may have existed in an RNA
world present before protein.
Two transesterifications characterize the mechanism in which
group I introns are spliced:

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1. 3'OH of a free guanine nucleoside (or one located in the

intron) or a nucleotide cofactor (GMP, GDP, GTP) attacks
phosphate at the 5' splice site.
2. 3'OH of the 5' exon becomes a nucleophile and the
second transesterification results in the joining of the
two exons.
The mechanism in which group II introns are spliced (two
transesterification reaction like group I introns) is as follows:

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1. The 2'OH of a specific adenosine in the intron attacks

the 5' splice site, thereby forming the lariat.

2. The 3'OH of the 5' exon triggers the second

transesterification at the 3' splice site, thereby joining
the exons together.

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Spliceosome mediated mechanism:

Spliceosomal complex:
Introns:
The word intron is derived from the terms intragenic
region, and intracistron, that is, a segment of DNA that is
located between two exons of a gene. The term intron refers to
both the DNA sequence within a gene and the corresponding
sequence in the unprocessed RNA transcript. As part of the

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RNA processing pathway, introns are removed by RNA splicing

either shortly after or concurrent with transcription. Introns
are found in the genes of most organisms and many viruses.
They can be located in a wide range of genes, including those
that generate proteins, ribosomal RNA (rRNA), and transfer
RNA (tRNA).

Within introns, a donor site (5' end of the intron), a branch site
(near the 3' end of the intron) and an acceptor site (3' end of
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the intron) are required for splicing. The splice donor site
includes an almost invariant sequence GU at the 5' end of the
intron, within a larger, less highly conserved region. The splice
acceptor site at the 3' end of the intron terminates the intron
with an almost invariant AG sequence. Upstream (5'-ward)
from the AG there is a region high in pyrimidines (C and U),
or polypyrimidine tract. Further upstream from the
polypyrimidine tract is the branchpoint, which includes an
adenine nucleotide involved in lariat formation. The consensus
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sequence for an intron (in IUPAC nucleic acid notation) is: G-G-
[cut]-G-U-R-A-G-U (donor site) ... intron sequence ... Y-U-R-A-C
(branch sequence 20-50 nucleotides upstream of acceptor
site) ... Y-rich-N-C-A-G-[cut]-G (acceptor site). However, it is
noted that the specific sequence of intronic splicing elements
and the number of nucleotides between the branchpoint and
the nearest 3’ acceptor site affect splice site selection. Also,
point mutations in the underlying DNA or errors during
transcription can activate a cryptic splice site in part of the
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transcript that usually is not spliced. This results in a mature

messenger RNA with a missing section of an exon. In this way,
a point mutation, which might otherwise affect only a single
amino acid, can manifest as a deletion or truncation in the final
protein.

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Formation and activity:

Splicing is catalyzed by the spliceosome, a large RNA-protein
complex composed of five small nuclear ribonucleoproteins
(snRNPs). Assembly and activity of the spliceosome occurs
during transcription of the pre-mRNA. The RNA components
of snRNPs interact with the intron and are involved in
catalysis. Two types of spliceosomes have been identified
(major and minor) which contain different snRNPs.

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Spliceosome:

 A spliceosome is a large and complex molecular machine

found primarily within the nucleus of eukaryotic cells.
 The spliceosome is assembled from small nuclear RNAs
(snRNA) and approximately 80 proteins.
 The spliceosome removes introns from a transcribed pre-
mRNA, a type of primary transcript.

 This process is generally referred to as splicing.

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 The major spliceosome splices introns containing GU at the

5' splice site and AG at the 3' splice site. It is composed of
the U1, U2, U4, U5, and U6 snRNPs and is active in the
nucleus. In addition, a number of proteins including U2 small
nuclear RNA auxiliary factor
1 (U2AF35), U2AF2 (U2AF65) and SF1 are required for the
assembly of the spliceosome. The spliceosome forms
different complexes during the splicing process:

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 Complex E
o The U1 snRNP binds to the GU sequence at the 5' splice
site of an intron;
o Splicing factor 1 binds to the intron branch point
sequence;
o U2AF1 binds at the 3' splice site of the intron;
o U2AF2 binds to the polypyrimidine tract;

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 Complex A (pre-spliceosome)
o The U2 snRNP displaces SF1 and binds to the branch
point sequence and ATP is hydrolyzed;

 Complex B (pre-catalytic spliceosome)

o The U5/U4/U6 snRNP trimer binds, and the U5 snRNP
binds exons at the 5' site, with U6 binding to U2;

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 Complex B*
o The U1 snRNP is released, U5 shifts from exon to intron,
and the U6 binds at the 5' splice site;

 Complex C (catalytic spliceosome)

o U4 is released, U6/U2 catalyzes transesterification,
making the 5'-end of the intron ligate to the A on intron
and form a lariat, U5 binds exon at 3' splice site, and the
5' site is cleaved, resulting in the formation of the lariat;

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 Complex C* (post-spliceosomal complex)

o U2/U5/U6 remain bound to the lariat, and the 3' site is
cleaved and exons are ligated using ATP hydrolysis. The
spliced RNA is released, the lariat is released and
degraded, and the snRNPs are recycled.

This type of splicing is termed canonical splicing or termed

the lariat pathway, which accounts for more than 99% of
splicing. By contrast, when the intronic flanking sequences
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do not follow the GU-AG rule, noncanonical splicing is said

to occur.

 The minor spliceosome is very similar to the major

spliceosome, but instead it splices out rare introns with
different splice site sequences. While the minor and major
spliceosomes contain the same U5 snRNP, the minor
spliceosome has different but functionally analogous snRNPs
for U1, U2, U4, and U6, which are respectively
called U11, U12, U4atac, and U6atac.
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Alternative splicing:
Alternative splicing is a method cells use to create many
proteins from the same strand of DNA. It is also called
alternative RNA splicing.

Alternative splicing, or alternative RNA splicing, or differential

splicing, is a regulated process during gene expression that
results in a single gene coding for multiple proteins. In this
process, particular exons of a gene may be included within or
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excluded from the final, processed messenger RNA (mRNA)

produced from that gene. Thus, the proteins translated from
alternatively spliced mRNAs will contain differences in their
amino acid sequence and, often, in their biological functions.

In regular DNA translation, specialized proteins create

messenger RNA (mRNA) from the DNA template. This mRNA
then finds its way to a ribosome, where the RNA code is

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MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA
 COMPILED AND CIRCULATED BY DR. POULAMI ADHIKARY MUKHERJEE, ASSISTANT
PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

translated into the structure of a new protein. In alternative

splicing, interactions between different proteins, the cell, and
the environment can cause different segments of the original
DNA to be omitted from the mRNA. When this happens, the
alternate mRNA is translated into an entirely different protein.

Proteins differ only in the basic arrangement of their amino

acids, which is dictated by the mRNA. Once that is changed, the
function of the protein changes. Using the method of
ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL
MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA
 COMPILED AND CIRCULATED BY DR. POULAMI ADHIKARY MUKHERJEE, ASSISTANT
PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

alternative splicing, organisms can produce many more

proteins than their DNA might indicate. For instance, humans
have around 20,000 genes which code for a protein. However,
there are thought to be over 100,000 different proteins in the
human body. Alternative splicing creates these different forms.

Alternative splicing occurs as a normal phenomenon in

eukaryotes, where it greatly increases the biodiversity of
proteins that can be encoded by the genome; in humans,
ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL
MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA
 COMPILED AND CIRCULATED BY DR. POULAMI ADHIKARY MUKHERJEE, ASSISTANT
PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

~95% of multi-exonic genes are alternatively spliced. There

are numerous modes of alternative splicing observed, of which
the most common is exon skipping. In this mode, a particular
exon may be included in mRNAs under some conditions or in
particular tissues, and omitted from the mRNA in others.

The production of alternatively spliced mRNAs is regulated by

a system of trans-acting proteins that bind to cis-acting sites
on the primary transcript itself. Such proteins include splicing
ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL
MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA
 COMPILED AND CIRCULATED BY DR. POULAMI ADHIKARY MUKHERJEE, ASSISTANT
PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

activators that promote the usage of a particular splice site,

and splicing repressors that reduce the usage of a particular
site. Mechanisms of alternative splicing are highly variable,
and new examples are constantly being found, particularly
through the use of high-throughput techniques.

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MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA
 COMPILED AND CIRCULATED BY DR. POULAMI ADHIKARY MUKHERJEE, ASSISTANT
PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL

MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA
 COMPILED AND CIRCULATED BY DR. POULAMI ADHIKARY MUKHERJEE, ASSISTANT
PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

Modes:
Five basic modes of alternative splicing are generally
recognized.

Exon skipping or cassette exon: in this case, an exon may be

spliced out of the primary transcript or retained. This is the
most common mode in mammalian pre-mRNAs.

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MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA
 COMPILED AND CIRCULATED BY DR. POULAMI ADHIKARY MUKHERJEE, ASSISTANT
PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

Mutually exclusive exons: One of two exons is retained in

mRNAs after splicing, but not both.

Alternative donor site: An alternative 5' splice junction (donor

site) is used, changing the 3' boundary of the upstream exon.

Alternative acceptor site: An alternative 3' splice junction

(acceptor site) is used, changing the 5' boundary of the
downstream exon.

ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL

MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA
 COMPILED AND CIRCULATED BY DR. POULAMI ADHIKARY MUKHERJEE, ASSISTANT
PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

Intron retention: A sequence may be spliced out as an intron

or simply retained. This is distinguished from exon skipping
because the retained sequence is not flanked by introns. If the
retained intron is in the coding region, the intron must encode
amino acids in frame with the neighboring exons, or a stop
codon or a shift in the reading frame will cause the protein to
be non-functional. This is the rarest mode in mammals.

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MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA
 COMPILED AND CIRCULATED BY DR. POULAMI ADHIKARY MUKHERJEE, ASSISTANT
PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

In addition to these primary modes of alternative splicing,

there are two other main mechanisms by which different
mRNAs may be generated from the same gene; multiple
promoters and multiple polyadenylation sites. Use of multiple
promoters is properly described as a transcriptional
regulation mechanism rather than alternative splicing; by
starting transcription at different points, transcripts with
different 5'-most exons can be generated. At the other end,
multiple polyadenylation sites provide different 3' end points
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MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA
 COMPILED AND CIRCULATED BY DR. POULAMI ADHIKARY MUKHERJEE, ASSISTANT
PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

for the transcript. Both of these mechanisms are found in

combination with alternative splicing and provide additional
variety in mRNAs derived from a gene.

Schematic cutoff from 3 splicing structures in the murine

hyaluronidase gene. Directionality of transcription from 5' to
3' is shown from left to right. These modes describe basic
splicing mechanisms, but may be inadequate to describe
complex splicing events. For instance, the figure to the right
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MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA
 COMPILED AND CIRCULATED BY DR. POULAMI ADHIKARY MUKHERJEE, ASSISTANT
PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

shows 3 spliceforms from the mouse hyaluronidase 3 gene.

Comparing the exonic structure shown in the first line (green)
with the one in the second line (yellow) shows intron
retention, whereas the comparison between the second and
the third spliceform (yellow vs. blue) exhibits exon skipping. A
model nomenclature to uniquely designate all possible splicing
patterns has recently been proposed.

ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL

MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA
 COMPILED AND CIRCULATED BY DR. POULAMI ADHIKARY MUKHERJEE, ASSISTANT
PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL

MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA
 COMPILED AND CIRCULATED BY DR. POULAMI ADHIKARY MUKHERJEE, ASSISTANT
PROFESSOR, DEPARTMENT OF ZOOLOGY, NARAJOLE RAJ COLLEGE

THA N K Y O U

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MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA