RNA Splicing- Definition, process, mechanism, types, errors, uses

RNA splicing is a process in molecular biology that transforms a newly-made precursor messenger RNA (pre-mRNA) transcript into a mature messenger RNA (mRNA) that can be translated into a protein. RNA splicing works by removing all the introns (non-coding regions of RNA) and splicing back together exons (coding regions).

RNA splicing is essential for gene expression and protein diversity in eukaryotic cells, where most genes contain introns that interrupt the coding sequences. RNA splicing occurs in the nucleus either during or immediately after transcription. For nuclear-encoded genes, splicing is usually required to create an mRNA molecule that can be exported to the cytoplasm and recognized by the ribosomes.

RNA splicing is catalyzed by a large and complex molecule called the spliceosome, which consists of five small nuclear RNAs (snRNAs) and about 80 protein molecules. The spliceosome recognizes the splice sites at the 5` and 3` ends of the introns and performs two transesterification reactions that result in the excision of the intron and the ligation of the exons.

Besides the spliceosome-mediated splicing, there are other types of RNA splicing that occur in nature, such as self-splicing and tRNA splicing. Self-splicing is a type of RNA splicing that occurs in some rare introns that can catalyze their own removal from the pre-mRNA without the help of other proteins or spliceosomes. tRNA splicing is a type of RNA splicing that occurs in tRNA genes that are interrupted by introns, but involves a different mechanism and enzymes than spliceosomal splicing.

Another important aspect of RNA splicing is alternative splicing, which is a process that allows a single pre-mRNA to be spliced in different ways, resulting in multiple mRNA variants that encode different proteins. Alternative splicing is a major source of protein diversity and complexity in eukaryotic cells, and it also plays a vital role in cellular differentiation and organism development.

RNA splicing is a highly regulated and precise process that affects many biological functions and pathways. However, errors or mutations in RNA splicing can lead to various diseases, such as cancer, neurodegenerative disorders, and genetic syndromes.

Introns are non-coding DNA sequences present within a gene that are removed by the process of RNA splicing during maturation of the RNA transcript. The word ‘introns’ is used to denote both the DNA sequences within the gene and the corresponding sequence in RNA transcripts.

Introns are common in the protein-coding nuclear genes of most eukaryotic organisms, as well as some unicellular organisms like bacteria. However, the number and length of introns vary widely among different species and genes. For example, the human genome contains about 190,000 introns, with an average length of 3,363 base pairs (bp), while the yeast genome has only 295 introns, with an average length of 246 bp.

Introns have a donor site (5′ end), a branch site (near the 3′ end), and an acceptor site (3′ end) that are required for splicing. These sites contain specific nucleotide sequences that are recognized by the splicing machinery. The donor site usually has a GU dinucleotide at the 5′ end of the intron, followed by an AG dinucleotide at the 3′ end of the exon. The branch site is located 18-40 nucleotides upstream of the acceptor site and has a conserved adenine (A) nucleotide. The acceptor site usually has an AG dinucleotide at the 3′ end of the intron, preceded by a pyrimidine-rich region.

Introns are crucial because they provide opportunities for genetic variation and regulation. For instance, introns can undergo alternative splicing, which is a process that produces different combinations of exons in the mature RNA transcript. This results in different protein isoforms with distinct functions and interactions. Alternative splicing is involved in many biological processes, such as development, differentiation, and disease.

Introns can also contain regulatory elements that influence gene expression. These elements can act as enhancers or silencers that modulate the transcription of nearby genes. They can also affect the splicing efficiency and accuracy of other introns or exons. Moreover, introns can encode small non-coding RNAs (such as microRNAs and snoRNAs) that have various roles in gene regulation.

Introns are not simply junk DNA, but rather dynamic and functional components of the genome that contribute to its complexity and diversity.

Exons are protein-coding DNA sequences that contain the necessary codons or genetic information essential for protein synthesis . The word ‘exon’ represents the expressed region present in the genome. The exosome is the term used to indicate the entire set of all exons present in the genome of the organisms.

Exons are found in all organisms ranging from jawed vertebrates to yeasts, bacteria, and even viruses. In the human genome, exons account for only 1% of the total genome while the rest is occupied by intergenic DNA and introns. Exons are essential units in protein synthesis as they carry regions composed of codons that code for various proteins.

Alternative splicing enables exons to be arranged in different combinations, where different configuration results in different proteins. A process similar to alternative splicing is exon shuffling where exons or sister chromosomes are exchanged during recombination.

Exons can refer to both the DNA and RNA sequences. Exons are separated by introns, which are non-coding sequences that are removed by RNA splicing . Exons are transcribed to messenger RNA (mRNA), which then undergoes translation to produce proteins .

Exons are crucial for the diversity and evolution of proteins, as well as for the regulation of gene expression and cellular functions. Exons also have roles in various biological processes such as DNA repair, chromatin remodeling, and gene silencing. Exons are also involved in various diseases and disorders caused by mutations or aberrant splicing.

A spliceosome is a large and complex molecule formed of RNAs and proteins that regulate the process of RNA splicing. The spliceosome is composed of five small nuclear RNAs (snRNA) and about 80 protein molecules.

The combination of RNAs with these proteins results in the formation of an RNA-protein complex termed as small nuclear ribonucleoproteins (snRNPs). These are mostly confined within the nucleus where they remain associated with the immature pre-RNA transcripts.

These spliceosomes, in addition to working on RNA-RNA interactions, are also involved in RNA-protein interactions. The spliceosome functions as an editor that selectively cuts out unnecessary and incorrect materials (introns) to produce a functional final-cut.

All spliceosomes are involved in both the removal of introns and the ligation of remaining exons. Another set of spliceosomes termed ‘minor spliceosomes’ are also found in eukaryotic cells which have less abundant RNAs and are involved in the splicing of a rare class of pre-mRNA introns.

The spliceosome is assembled from small nuclear RNAs (snRNA) and a range of associated protein factors. When these small RNAs are combined with the protein factors, they make RNA-protein complexes called snRNPs (small nuclear ribonucleoproteins, pronounced “snurps”). The snRNAs that make up the major spliceosome are named U1, U2, U4, U5, and U6, so-called because they are rich in uridine, and participate in several RNA-RNA and RNA-protein interactions.

The assembly of the spliceosome occurs on each pre-mRNA at each exon:intron junction. The pre-mRNA introns contains specific sequence elements that are recognized and utilized during spliceosome assembly. These include the 5` end splice site, the branch point sequence, the polypyrimidine tract, and the 3` end splice site. The spliceosome catalyzes the removal of introns, and the ligation of the flanking exons.

The spliceosome undergoes several conformational changes and compositional rearrangements during the splicing process. The splicing cycle can be divided into four major stages: complex E (early), complex A (pre-spliceosome), complex B (spliceosome), and complex C (post-spliceosome).

Complex E is formed by the recognition of the 5` end splice site by U1 snRNP and other factors. Complex A is formed by the recruitment of U2 snRNP to the branch point sequence. Complex B is formed by the addition of U4/U6.U5 tri-snRNP to complex A. Complex C is formed by the activation of complex B for catalysis through the release of U1 and U4 snRNPs.

The catalytic steps of splicing involve two transesterification reactions that result in the cleavage of the 5` and 3` splice sites and the joining of the exons. The first reaction forms a lariat structure between the 5` end of the intron and the branch point adenosine. The second reaction releases the lariat intron and ligates the exons.

The released intron is then degraded by various mechanisms, while the spliced mRNA is exported to the cytoplasm for translation.

RNA splicing is the process of removing introns and joining exons from the pre-mRNA in the nucleus before it is translated into protein in the cytoplasm. RNA splicing involves two main steps: cutting the pre-mRNA at the 5′ and 3′ splice sites and joining the exons together. RNA splicing is catalyzed by spliceosomes, which are complexes of small nuclear ribonucleoproteins (snRNPs) that bind to the introns and the exons. RNA splicing occurs by transesterification, which is a chemical reaction that involves the exchange of hydroxyl (OH) groups between RNA nucleotides.

The process of splicing occurs in several steps. The RNA splicing steps are:

• Step 1: Formation and activation of different spliceosome complexes

  • The spliceosome consists of five snRNPs: U1, U2, U4, U5, and U6. Each snRNP contains one or more snRNAs and several proteins. The snRNAs base-pair with each other and with the pre-mRNA to form a three-dimensional structure that facilitates splicing.
  • The first step of splicing is the recognition and binding of U1 snRNP to the 5′ splice site of the intron. This is followed by the binding of U2 snRNP to a conserved branch point region within the intron, where an adenine nucleotide forms a bulge. The branch point is essential for lariat formation in the next step.
  • The U4/U6.U5 tri-snRNP then joins the complex, bringing together the 5′ and 3′ splice sites. The U4 snRNP masks the activity of U6 snRNP, which will act as a catalyst in the subsequent steps. The spliceosome is now fully assembled and ready for splicing.

• Step 2: Finding the starting and ending points of the introns and removing them

  • The second step of splicing is the cleavage of the 5′ splice site and the formation of a lariat structure. This is achieved by a transesterification reaction, in which the 2′ OH group of the branch point adenine attacks the phosphodiester bond at the 5′ splice site, resulting in a covalent bond between the intron and the branch point. The 5′ end of the intron is released from the exon and forms a loop.
  • The third step of splicing is the cleavage of the 3′ splice site and the ligation of the exons. This is also achieved by a transesterification reaction, in which the free 3′ OH group of the upstream exon attacks the phosphodiester bond at the 3′ splice site, resulting in a covalent bond between the exons. The intron is released as a lariat structure that will be degraded by nucleases.

• Step 3: Joining the exons together

  • The final step of splicing is the release of the mature mRNA from the spliceosome. The snRNPs dissociate from each other and from the mRNA, recycling for another round of splicing. The mRNA is then exported to the cytoplasm for translation.

The process of RNA splicing can be summarized by this equation:

pre-mRNA + nATP + nGTP + nCTP + nUTP → mRNA + nPPi + intron lariat

where n represents an integer number.

RNA splicing is the process of removing introns and joining exons in a pre-mRNA transcript. There are different types of RNA splicing that occur in different organisms and under different conditions. Some of the major types of RNA splicing are:

• Self-splicing: This is a type of RNA splicing that occurs in some rare introns that can catalyze their own removal and exon ligation without the help of any proteins or spliceosomes . These introns act as ribozymes, which are RNA molecules with enzymatic activity. There are three groups of self-splicing introns: Group I, Group II, and Group III. Group I and Group II introns follow a similar mechanism to spliceosomes, involving two transesterification reactions and a lariat intermediate. Group III introns are found only in bacteriophages and use a hydrolytic mechanism.
• Alternative splicing: This is a type of RNA splicing that generates multiple mRNA variants from the same pre-mRNA transcript by using different combinations of exons . Alternative splicing is a major source of protein diversity and complexity in eukaryotes, as it allows one gene to encode for different proteins with different functions. Alternative splicing is regulated by various factors, such as cis-acting elements (sequences in the pre-mRNA that affect splicing) and trans-acting factors (proteins or RNAs that bind to the pre-mRNA and influence splicing). Alternative splicing can also be influenced by external stimuli, such as stress, hormones, or diseases.

• tRNA splicing: This is a type of RNA splicing that occurs in tRNA transcripts, which are also interrupted by introns. However, tRNA splicing differs from mRNA splicing in several aspects. First, tRNA splicing is catalyzed by three enzymes: an endonuclease, a ligase, and a phosphotransferase . Second, tRNA splicing requires ATP hydrolysis for energy. Third, tRNA splicing occurs in both the nucleus and the cytoplasm. Fourth, tRNA splicing involves the transfer of the phosphate group from the 3` end of the intron to NAD+, forming cyclic NAD+.

  RNA Splicing errors

RNA splicing is a precise and regulated process that ensures the correct expression of most eukaryotic genes. However, errors in splicing can occur due to mutations or environmental factors that affect the recognition of splice sites, the assembly of spliceosomes, or the activity of splicing factors. RNA splicing errors can result in various splicing-related diseases, such as muscular dystrophies, neurodegenerative disorders, cancers, and autoimmune diseases.

Some of the common types of RNA splicing errors are:

• Exon skipping: This occurs when an exon is skipped and not included in the mature mRNA. This can cause a frameshift mutation or a premature stop codon, leading to a truncated or nonfunctional protein. Exon skipping can be caused by mutations that weaken the splice sites or disrupt the binding of splicing enhancers. An example of a disease caused by exon skipping is Duchenne muscular dystrophy (DMD), which results from the absence of exon 44 in the dystrophin gene.
• Intron retention: This occurs when an intron is not removed and remains in the mature mRNA. This can also cause a frameshift mutation or a premature stop codon, or interfere with the translation or stability of the mRNA. Intron retention can be caused by mutations that strengthen the splice sites or disrupt the binding of splicing silencers. An example of a disease caused by intron retention is spinal muscular atrophy (SMA), which results from the retention of intron 7 in the SMN2 gene.
• Alternative splicing: This occurs when different combinations of exons are included or excluded from the mature mRNA, resulting in different protein isoforms. Alternative splicing is a normal and regulated process that increases the diversity and complexity of gene expression. However, aberrant alternative splicing can occur due to mutations or dysregulation of splicing factors, leading to abnormal protein isoforms that may have altered functions or interactions. An example of a disease caused by aberrant alternative splicing is amyotrophic lateral sclerosis (ALS), which results from the inclusion of an extra exon in the FUS gene.
• Cryptic splice site activation: This occurs when a mutation creates a new splice site that competes with the original splice site, resulting in the inclusion or exclusion of an extra sequence in the mature mRNA. This can cause a frameshift mutation or a change in the protein domain structure, affecting its function or interaction. An example of a disease caused by cryptic splice site activation is atypical progeria syndrome (APS), which results from the activation of a cryptic splice site in intron 11 of the LMNA gene.

RNA splicing errors can have various consequences on cellular functions, such as:

• Loss of function: This occurs when the splicing error leads to a reduced or absent expression of a functional protein, resulting in impaired cellular processes. For example, exon skipping in DMD causes a loss of function of dystrophin, a protein that stabilizes muscle fibers.
• Gain of function: This occurs when the splicing error leads to an increased or novel expression of a functional protein, resulting in enhanced or aberrant cellular processes. For example, alternative splicing in FUS causes a gain of function of FUS, a protein that regulates RNA metabolism and forms toxic aggregates in neurons.
• Dominant negative effect: This occurs when the splicing error leads to an expression of a mutant protein that interferes with the function of the normal protein, resulting in reduced or altered cellular processes. For example, cryptic splice site activation in LMNA causes a dominant negative effect of progerin, a mutant form of lamin A that disrupts nuclear structure and function.

RNA splicing errors can be detected by various methods, such as:

• RNA sequencing: This is a technique that sequences and analyzes RNA molecules from a sample, allowing the identification and quantification of different splice variants and their expression levels.
• Reverse transcription polymerase chain reaction (RT-PCR): This is a technique that converts RNA molecules into complementary DNA (cDNA) and amplifies them by PCR, allowing the detection and measurement of specific splice variants by gel electrophoresis or real-time PCR.
• Splice site prediction tools: These are computational tools that use algorithms and databases to predict potential splice sites and their strength based on sequence features and conservation.

RNA splicing errors can be corrected by various strategies, such as:

• Antisense oligonucleotides (ASOs): These are synthetic nucleic acid molecules that bind to specific RNA sequences and modulate their splicing by blocking or enhancing splice sites or splicing factors. For example, eteplirsen is an ASO that induces exon skipping in DMD patients to restore dystrophin expression.
• Small molecule modulators: These are chemical compounds that bind to specific RNA sequences or proteins and modulate their splicing by altering their structure or activity. For example, risdiplam is a small molecule modulator that enhances exon inclusion in SMA patients to increase SMN2 expression.
• Gene therapy: This is a technique that delivers functional genes into cells using viral or non-viral vectors, allowing the expression of normal proteins or RNA molecules that can correct splicing defects. For example, Zolgensma is a gene therapy that delivers SMN1 gene into SMA patients using adeno-associated virus vectors.

RNA splicing errors are important sources of genetic variation and disease pathogenesis. Understanding their mechanisms and consequences can help to develop novel diagnostic and therapeutic approaches for various human disorders.

Applications of RNA Splicing

RNA splicing is a process that removes introns and joins exons in pre-mRNA transcripts, resulting in mature mRNA that can be translated into proteins. RNA splicing has various biological and medical applications, some of which are:

• RNA splicing facilitates the formation of multiple functional mRNAs from a single transcript, which codes for different proteins. This increases the diversity and complexity of gene expression and protein function in eukaryotic cells.
• RNA splicing also helps in the regulation of gene expression and protein content of the cell. Splicing can be influenced by various factors, such as splice site strength, trans-acting factors, chromatin structure, RNA structure, and alternative transcription initiation or termination. Splicing can affect the stability, localization, interactions, and activity of mRNA and proteins.
• RNA splicing assists in the evolution process by forming different combinations of exons and thereby making new and improved proteins. Splicing can also generate new splice sites or remove existing ones by mutations, resulting in novel exons or introns. Splicing can also facilitate exon shuffling, which is the exchange of exons between genes during recombination.
• RNA splicing has a role in many human diseases, especially cancers. Aberrant splicing can result from mutations in splice sites, splicing factors, or regulatory elements, leading to the production of defective or harmful proteins. Alternatively spliced isoforms can also act as markers for cancer diagnosis and prognosis, as well as targets for cancer therapy. For example, some splice variants of the Bcl-2 gene are associated with apoptosis resistance in cancer cells.
• RNA splicing can be used as a therapeutic strategy to correct mutations at the mRNA level or to induce cell death in target cells. For example, trans-splicing is a type of splicing that joins exons from different pre-mRNA transcripts to generate a chimeric product. Trans-splicing can be used to repair mutations by replacing defective exons with normal ones or to introduce toxins, apoptotic factors, or suicide genes into cancer cells.

RNA splicing is a process that removes introns and joins exons in pre-mRNA transcripts, resulting in mature mRNA that can be translated into proteins. RNA splicing has various biological and medical applications, some of which are:

• RNA splicing facilitates the formation of multiple functional mRNAs from a single transcript, which codes for different proteins. This increases the diversity and complexity of gene expression and protein function in eukaryotic cells.
• RNA splicing also helps in the regulation of gene expression and protein content of the cell. Splicing can be influenced by various factors, such as splice site strength, trans-acting factors, chromatin structure, RNA structure, and alternative transcription initiation or termination. Splicing can affect the stability, localization, interactions, and activity of mRNA and proteins.
• RNA splicing assists in the evolution process by forming different combinations of exons and thereby making new and improved proteins. Splicing can also generate new splice sites or remove existing ones by mutations, resulting in novel exons or introns. Splicing can also facilitate exon shuffling, which is the exchange of exons between genes during recombination.
• RNA splicing has a role in many human diseases, especially cancers. Aberrant splicing can result from mutations in splice sites, splicing factors, or regulatory elements, leading to the production of defective or harmful proteins. Alternatively spliced isoforms can also act as markers for cancer diagnosis and prognosis, as well as targets for cancer therapy. For example, some splice variants of the Bcl-2 gene are associated with apoptosis resistance in cancer cells.
• RNA splicing can be used as a therapeutic strategy to correct mutations at the mRNA level or to induce cell death in target cells. For example, trans-splicing is a type of splicing that joins exons from different pre-mRNA transcripts to generate a chimeric product. Trans-splicing can be used to repair mutations by replacing defective exons with normal ones or to introduce toxins, apoptotic factors, or suicide genes into cancer cells.

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