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> > >Unit 4: > > >Post Transcriptional Modifications, Processing of Eukaryotic RNA and
Gene Regulation > > >
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>Gene Splicing >
>Splicing is a key process in gene expression that involves removing introns
and joining exons to generate the mature mRNA. This is a highly regulated
process that takes place within the nucleus of eukaryotic cells and is
mediated by large and complex macromolecular machinery called
spliceosomes. >
>Gene Splicing Introduction >
>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 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. >
>Gene Splicing: Various forms of gene splicing >
>There are several types of common gene splicing events. These are the
events that can simultaneously occur in the 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 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. >
>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. >
>Splice Variant Detection Methods >
>Gene splicing leads to the synthesis of alternate proteins that play an
important role in the human physiology and disease. Currently, the most
efficient methods for large scale detection of splice variants include
computational prediction methods and microarray analysis. Microarray based
splice variant detection is the most popular method currently in use. The
highly parallel and sensitive nature of microarrays make them ideal for
monitoring gene expression on a tissue-specific, genome-wide level.
Microarray based methods for detecting splice variants provide a robust,
scalable platform for high-throughput discovery of alternative gene
splicing. A number of novel gene transcripts were detected using microarray
based methods that were not detected by ESTs using computational methods.
Another commonly used method for discovering of novel gene isoforms is
RT-PCR followed by sequencing. This is a powerful approach and can be
effectively used for analyzing a small number of genes. However, it only
provides only a limited view of the gene structure, is labor-intensive, and
does not easily scale to thousands of genes or hundreds of tissues. >
>Challenges in Microarray Design for Splice Variant Detection >
>Microarray based gene splicing detection poses some unique challenges in
designing probes for isoforms that show a high degree of homology. In order
to differentiate between these isoforms, a microarray that uses a
combination of probes for exons and exon-exon junctions is used. Exon
skipping events or other deletions can be monitored by using junction
probes. For example, a probe spanning the exon 1 and exon 3 of the gene will
detect the skipping of exon 2 from the gene that is translated into a
protein. >
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>Splicing takes place in two stages >
>The first step involves recognition and cleavage of the 5' and 3' splice
sites of introns, and the second step joins exons together to form the
mature mRNA. A detailed overview of the splicing mechanism follows. >
>Step 1: Detect and Cut Splices >
>The first step in splicing involves recognition and cleavage of splice
junctions flanking introns. The spliceosome complex recognizes these sites
by binding to specific sequences in the pre-mRNA located at the 5' and 3'
ends of introns. These sequences, called 5' and 3' splice sites,
respectively, usually consist of short consensus sequences that are highly
conserved between species. >
>Splice site recognition involves a series of interactions between the
spliceosome complex and the pre-mRNA to form the spliceosome complex.
Spliceosomes are large macromolecular machines composed of snRNAs (small
nuclear RNAs) and proteins. The snRNA functions as the catalytic subunit of
the spliceosome, and the protein provides structural support and helps
stabilize the complex. >
>Once assembled, the spliceosome complex cleaves the mRNA at the 5' and 3'
splice sites. This cleavage creates short RNA sequences called introns that
are removed from the pre-mRNA and degraded. >
>Step 2: Exon Ligation >
>The second step of splicing joins the exons to form the mature mRNA. This
process is carried out by a spliceosome complex that catalyzes the joining
of two exons. >
>After the intron is removed, the spliceosome complex undergoes a series of
conformational changes that join the two exons. A spliceosome bridge is
formed that spans the two exons, bringing the two exons into close
proximity. When the exons join, the spliceosome catalyzes the formation of
phosphodiester bonds between the two exons, joining them together to form
the mature mRNA. After the exons are ligated, the spliceosome is degraded
and the mature mRNA is transported from the nucleus to the cytoplasm and
translated into protein. >
>alternate splicing >
>An important aspect of splicing is alternative splicing, which can generate
multiple protein variants from a single gene. Alternative splicing occurs
when different combinations of exons are included or excluded in the mature
mRNA. This is achieved by using alternative splice sites within the pre-mRNA
that can be recognized by the spliceosome complex to generate different mRNA
isoforms. >
>Alternative splicing is a highly regulated process that is controlled by a
variety of factors, including the sequence of the pre-mRNA, the availability
of spliceosome components, and the presence of regulatory proteins that can
bind to the pre-mRNA and modulate splicing. >
>Conclusion >
>Splicing is a critical process in gene expression that is responsible for
the removal of introns and the joining together of exons to produce mature
mRNA. This process is highly regulated and occurs in the nucleus of
eukaryotic cells, where it is mediated by a large and complex macromolecular
machine called the spliceosome. >
>Alternative splicing >
>Alternative splicing is the process by which different combinations of
exons within the pre-mRNA molecule are spliced together to generate
multiple mRNA transcripts, each of which can be translated into a different
protein. In other words, a single gene can generate multiple protein
isoforms with different structures and functions by altering the way
pre-mRNA is spliced. >
>In alternative splicing, the pre-mRNA molecule is processed by a
spliceosome, a complex of RNA and protein that removes introns and joins
exons. Depending on the specific combination of splice sites recognized by
the spliceosome, different exons are included or excluded from the final
mRNA transcript, giving rise to different protein isoforms. >
>Alternative splicing is a common mechanism of gene regulation in
eukaryotes, allowing increased proteomic diversity and specialization of
cellular functions. It is estimated that up to 95% of human genes are
alternatively spliced. >