Exon- Definition, Structure, Splicing, Process of Splicing

An exon is a segment of DNA or RNA that codes for a part of a protein or a functional RNA molecule. Exons are found in the genes of eukaryotes, which are organisms with a nucleus and other membrane-bound organelles. Exons are separated by non-coding regions called introns, which are removed during the process of RNA splicing.

Exons can vary in size and number depending on the gene and the organism. In humans, the average exon length is about 170 base pairs (bp), and the average number of exons per gene is about 9. However, some exons can be as short as 2 bp or as long as 11,555 bp, and some genes can have more than 100 exons.

Exons have different roles and functions depending on their location and sequence. Exons can be classified into three types:

• Coding exons contain the nucleotide triplets that specify the amino acids of a protein. Coding exons usually start with a start codon (AUG) and end with a stop codon (UAA, UAG, or UGA).
• Untranslated exons are located at the 5` and 3` ends of a messenger RNA (mRNA) molecule and do not code for any amino acids. Untranslated exons regulate the stability, transport, and translation of the mRNA.
• Non-coding exons are found in genes that produce non-coding RNAs, such as ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and long non-coding RNA (lncRNA). Non-coding exons may have structural or regulatory functions in these RNAs.

The figure below shows an example of a gene with three coding exons (red) and two untranslated exons (grey). The introns (blue) are spliced out to form a mature mRNA that can be translated into a protein.

Figure 1: Exon structure in a protein-coding gene.

Splicing is defined as the process in which introns, the non-coding regions, are excised out of the primary mRNA, and exons are joined together in the primary transcript. In this process, a pre-mRNA is formed into a mature mRNA.

Splicing occurs before the process of translation, before protein synthesis. It is an important process because correct protein cannot be coded without splicing. It also plays an important role in the regulation of gene expression and proteins.

Splicing allows for the generation of different protein isoforms from a single gene by alternative splicing. This increases the diversity and complexity of the proteome and enables cells to respond to different stimuli and conditions.

Splicing also affects the stability, localization, and interactions of mRNA molecules. Splicing can modulate the degradation of mRNA by affecting the recognition of decay signals or by exposing or hiding miRNA binding sites. Splicing can also influence the transport and localization of mRNA by affecting the binding of RNA-binding proteins or by creating or removing localization signals. Splicing can also alter the interactions of mRNA with other molecules, such as ribosomes, translation factors, or other RNAs.

Splicing is a highly regulated process that depends on various factors, such as the sequence and structure of the pre-mRNA, the availability and activity of splicing factors, and the cellular environment. Splicing can be affected by mutations, diseases, drugs, and stress.

Splicing is essential for normal development and function of eukaryotic organisms. Defects in splicing can cause various diseases, such as cancer, neurodegeneration, muscular dystrophy, and immunodeficiency.

Splicing is a complex and dynamic process that involves multiple steps and components. The main component of splicing is the spliceosome, a large RNA-protein complex that catalyzes the splicing reactions. The next point will describe the process of splicing and the role of spliceosome in detail.

Splicing is the process of removing introns, the non-coding regions, from the pre-mRNA and joining the exons, the coding regions, to form a mature mRNA. Splicing is catalyzed by a large RNA-protein complex called the spliceosome.

The spliceosome consists of five small nuclear ribonucleoproteins (snRNPs) and additional proteins that recognize the splice sites and the branch point of the intron. The snRNPs are named U1, U2, U4, U5 and U6, and each contains a small nuclear RNA (snRNA) and a protein component.

The process of splicing can be summarized as follows:

1. The U1 snRNP binds to the 5` splice site of the pre-mRNA, which has a conserved GU sequence.
2. The U2 snRNP binds to the branch point of the intron, which has an invariant A nucleotide.
3. The U4/U6 and U5 snRNPs join the complex, forming a complete spliceosome. The U5 snRNP bridges the two exons that will be joined together.
4. The U4 snRNP dissociates from the complex, allowing the U6 snRNA to interact with the U2 snRNA and the 5` splice site.
5. The 5` end of the intron is cleaved and attached to the branch point A, forming a lariat structure.
6. The 3` end of the intron is cleaved and the intron is released as a lariat. The two exons are ligated together by a phosphodiester bond.
7. The spliceosome disassembles and releases the spliced mRNA. The snRNPs are recycled for further splicing reactions.

The role of the spliceosome is not only to catalyze the splicing reaction, but also to regulate the splicing process and ensure its accuracy. The spliceosome can recognize different types of introns and splice them accordingly. The spliceosome can also perform alternative splicing, which is the process of producing different mRNA variants from the same pre-mRNA by using different combinations of exons. Alternative splicing increases the diversity of proteins encoded by a single gene and plays an important role in gene expression regulation. Furthermore, the spliceosome can interact with other factors involved in transcription, RNA processing and translation, forming a network of molecular interactions that coordinate different steps of gene expression.

The splicing pathway is the sequence of events that leads to the removal of introns and the joining of exons in the pre-mRNA. The splicing pathway involves the following steps:

• Recognition of splice sites: The splice sites are the specific sequences at the boundaries of introns and exons. The 5` splice site has a GU nucleotide pair, and the 3` splice site has an AG nucleotide pair. There is also a branch point within the intron, which is an adenine (A) nucleotide that forms a lariat structure with the 5` end of the intron. The splice sites are recognized by a complex of proteins and small nuclear RNAs (snRNAs) called the spliceosome.
• Formation of spliceosome: The spliceosome is composed of five small nuclear ribonucleoproteins (snRNPs), which are U1, U2, U4, U5, and U6. Each snRNP contains one or more snRNAs and several proteins. The snRNPs assemble on the pre-mRNA in a specific order. First, U1 binds to the 5` splice site, and U2 binds to the branch point. Then, U4/U6 and U5 join the complex, forming a complete spliceosome.
• Cleavage and ligation: The spliceosome catalyzes two transesterification reactions that result in the cleavage of the intron and the ligation of the exons. In the first reaction, the 5` end of the intron is cut and attached to the branch point A, forming a loop-like structure called a lariat. In the second reaction, the 3` end of the intron is cut and released, and the two exons are joined together by a phosphodiester bond. The spliced mRNA is then ready for export to the cytoplasm and translation.
• Recycling of spliceosome: After each splicing event, the spliceosome disassembles and releases its components. The snRNPs are recycled and reused for splicing other introns in the same or different pre-mRNAs. The intron lariat is degraded by nucleases in the nucleus.

The splicing pathway is highly regulated and precise, as any errors in splicing can lead to abnormal proteins or diseases. The splicing pathway can also generate diversity in gene expression by alternative splicing, which is the process of producing different mRNA variants from the same pre-mRNA by using different combinations of exons.

Alternative splicing is the process in which different variations in the mRNA are created by joining different exons. Alternative splicing leads to isoforms of proteins.

This means one gene can code for more than one type of mRNA, and more than one type of protein. This increases the diversity and complexity of the proteome, and allows for the regulation of gene expression and protein function.

The most common types of alternative splicing are:

• Exon skipping – in this process certain exons along with their adjacent introns are excised from the pre-mRNA before translation. This results in a shorter mRNA and a protein that lacks some amino acid sequences. Exon skipping is the most frequent type of alternative splicing in humans, and it can affect the structure and function of proteins. For example, exon skipping in the dystrophin gene causes Duchenne muscular dystrophy, a severe genetic disorder that affects muscle strength and movement.
• Alternative 5` splice site or 3` splice site – this can be achieved by joining of exons to alternative 3` or 5` splice sites. This results in a change in the length and sequence of the exons, and can affect the coding region or the untranslated region of the mRNA. Alternative splice sites can alter the binding affinity of splice factors, regulatory elements, or ribosomes, and can affect the stability, localization, or translation efficiency of the mRNA. For example, alternative splice sites in the insulin receptor gene can produce two isoforms of the receptor that have different binding affinities for insulin and different signaling pathways.
• Intron retention – this is achieved when some introns are retained in the mature mRNA. This results in a longer mRNA that contains non-coding sequences. Intron retention can introduce premature stop codons or alter the reading frame of the mRNA, leading to nonsense-mediated decay or frameshift mutations. Alternatively, intron retention can create new functional domains or interactions for the protein, or modulate its subcellular localization or degradation. For example, intron retention in the p53 gene can produce a truncated protein that acts as a dominant-negative inhibitor of the wild-type p53 protein, which is a tumor suppressor.
• Mutually exclusive exons – this is achieved when only one of two or more alternative exons is included in the mature mRNA. This results in a switch between different amino acid sequences in the protein. Mutually exclusive exons can generate proteins with different functional domains or interactions, or different substrate specificities or catalytic activities. For example, mutually exclusive exons in the tropomyosin gene can produce different isoforms of the protein that bind to different types of actin filaments and regulate muscle contraction.

These are some of the main types of alternative splicing that occur in eukaryotes. However, there are other less common types, such as alternative promoter usage, alternative polyadenylation site, or trans-splicing. Alternative splicing is a dynamic and regulated process that depends on various factors, such as splice factors, RNA-binding proteins, chromatin structure, transcription rate, cellular signals, or environmental stimuli.

Alternative splicing is an essential mechanism for generating protein diversity and complexity from a limited number of genes. It also plays an important role in various biological processes, such as development, differentiation, cell cycle, apoptosis, signal transduction, immune response, or neuronal function. Moreover, alternative splicing is involved in many human diseases, such as cancer, neurodegenerative disorders, cardiovascular diseases, or autoimmune diseases.

Self-splicing introns are a special type of introns that can remove themselves from the pre-mRNA without the help of spliceosome or any other proteins. They are also called ribozymes, because they act as both RNA and enzymes. Self-splicing introns are rare and mostly found in some organelles, such as mitochondria and chloroplasts, and some viruses and bacteria.

There are two main types of self-splicing introns: group I and group II. Both types use a similar mechanism of phosphoester transfer, but differ in their structure and catalytic sites.

Group I introns have a complex secondary structure with nine conserved domains (P1 to P9) that form a three-dimensional active site. The splicing reaction involves three steps:

1. A guanosine nucleotide (G) attacks the 5` splice site and cleaves the exon-intron junction, forming a 3` OH group on the exon and a 5` G-intron intermediate.
2. The free 3` OH group of the exon attacks the 3` splice site and cleaves the intron-exon junction, forming a 2` OH group on the intron and a 3` OH group on the exon.
3. The two exons are ligated together by a phosphodiester bond, releasing the intron as a linear molecule with a 2`-5` phosphodiester bond at the branch point.

Group II introns have a simpler secondary structure with six conserved domains (I to VI) that form a lariat-shaped active site. The splicing reaction involves two steps:

1. The 2` OH group of an adenine nucleotide (A) within the intron attacks the 5` splice site and cleaves the exon-intron junction, forming a 3` OH group on the exon and a 2`-5` A-intron intermediate.
2. The free 3` OH group of the exon attacks the 3` splice site and cleaves the intron-exon junction, forming a 3` OH group on the exon and releasing the intron as a lariat molecule with a 2`-5` phosphodiester bond at the branch point.

Self-splicing introns are important for understanding the evolution of RNA processing and catalysis. They may represent an ancient form of splicing that preceded the spliceosome. They may also have roles in gene regulation, genome rearrangement, and horizontal gene transfer.

Exons and splicing are essential for the expression of genes and the synthesis of proteins in eukaryotes. Exons are the segments of DNA or RNA that code for amino acids, the building blocks of proteins. Splicing is the process of removing introns, the non-coding regions, from the pre-mRNA and joining the exons together. Splicing ensures that only the relevant information is translated into proteins and that errors or mutations in the introns do not affect the protein function.

Splicing also allows for the generation of diversity and complexity in the proteome, the collection of proteins in a cell or organism. By using alternative splicing, different combinations of exons can be joined together to produce different variants of mRNA and proteins from the same gene. This increases the efficiency and versatility of gene expression and enables the regulation of protein activity and interactions in response to various signals and conditions.

Some RNA molecules can also perform self-splicing, which means they can remove their own introns without the help of spliceosomes or other proteins. This demonstrates the catalytic ability and evolutionary origin of RNA as a molecule that can store and manipulate genetic information.

In conclusion, exons and splicing are vital for the proper functioning of cells and organisms. They enable the accurate and diverse production of proteins that are involved in various biological processes and pathways. Any defects or abnormalities in exons or splicing can lead to diseases or disorders such as cancer, neurodegeneration, muscular dystrophy, and cystic fibrosis. Therefore, understanding the mechanisms and regulation of exons and splicing is important for advancing biomedical research and developing new therapies.

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