Alternative splicing
Encyclopedia
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
of a gene (a primary gene transcript or pre-mRNA) are reconnected in multiple ways during RNA splicing
RNA splicing
In molecular biology and genetics, splicing is a modification of an RNA after transcription, in which introns are removed and exons are joined. This is needed for the typical eukaryotic messenger RNA before it can be used to produce a correct protein through translation...
. The resulting different mRNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
s may be translated into different protein isoform
Protein isoform
A protein isoform is any of several different forms of the same protein. Different forms of a protein may be produced from related genes, or may arise from the same gene by alternative splicing. A large number of isoforms are caused by single-nucleotide polymorphisms or SNPs, small genetic...
s; thus, a single gene may code for multiple proteins.
Alternative splicing occurs as a normal phenomenon in eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s, where it is greatly increases the biodiversity of proteins that can be encoded by the genome; in humans, ~95% of multiexonic 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
Trans-acting
In the field of molecular biology, trans-acting , in general, means "acting from a different molecule"...
proteins that bind to cis-acting sites on the pre-mRNA itself. Such proteins include splicing 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. Researchers hope to fully elucidate the regulatory systems involved in splicing, so that alternative splicing products from a given gene under particular conditions could be predicted by a "splicing code".
Abnormal variations in splicing are also implicated in disease; a large proportion of human genetic disorder
Genetic disorder
A genetic disorder is an illness caused by abnormalities in genes or chromosomes, especially a condition that is present from before birth. Most genetic disorders are quite rare and affect one person in every several thousands or millions....
s result from splicing variants. Abnormal splicing variants are also thought to contribute to the development of cancer, although such aberrant splicing products are, under normal conditions, usually safeguarded and eliminated by a posttranscriptional quality control mechanism.
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. Both 5’ and 3’ splice sites varied, and in addition, the transcript contained multiple polyadenylationPolyadenylation
Polyadenylation is the addition of a poly tail to an RNA molecule. The poly tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA for translation...
sites, giving different 3’ ends for the processed mRNAs.
In 1981, the first example of alternative splicing in a transcript from a normal, endogenous gene was characterized. The gene encoding the thyroid
Thyroid
The thyroid gland or simply, the thyroid , in vertebrate anatomy, is one of the largest endocrine glands. The thyroid gland is found in the neck, below the thyroid cartilage...
hormone calcitonin
Calcitonin
Calcitonin is a 32-amino acid linear polypeptide hormone that is producedin humans primarily by the parafollicular cells of the thyroid, and in many other animals in the ultimobranchial body. It acts to reduce blood calcium , opposing the effects of parathyroid hormone . Calcitonin has been found...
was found to be alternatively spliced in mammalian cells. The pre-mRNA from this gene contains 6 exons; the calcitonin mRNA contains exons 1–4, and terminates after a polyadenylation site in exon 4. Another mRNA is produced from this pre-mRNA by skipping exon 4, and includes exons 1–3, 5, and 6. It encodes a protein known as CGRP (calcitonin gene related peptide). 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. The "record-holder" for alternative splicing is a D. melanogaster gene called Dscam
DSCAM
DSCAM and Dscam are both abbreviations for Down Syndrome Cell Adhesion Molecule. The case difference is significant: DSCAM refers to the human protein, and Dscam refers to its analog in Drosophila....
, which could potentially have 38,016 splice variants.
Modes
- 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.
- 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.
- 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 frameReading frameIn biology, a reading frame is a way of breaking a sequence of nucleotides in DNA or RNA into three letter codons which can be translated in amino acids. There are 3 possible reading frames in an mRNA strand: each reading frame corresponding to starting at a different alignment...
will cause the protein to be non-functional. This is the rarest mode in mammals.
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
Polyadenylation
Polyadenylation is the addition of a poly tail to an RNA molecule. The poly tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA for translation...
sites. Use of multiple promoters is properly described as a transcriptional regulation
Transcriptional regulation
Transcriptional regulation is the change in gene expression levels by altering transcription rates. -Regulation of transcription:Regulation of transcription controls when transcription occurs and how much RNA is created...
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 for the transcript. Both of these mechanisms are found in combination with alternative splicing and provide additional variety in mRNAs derived from a gene.
These modes describe basic splicing mechanisms, but may be inadequate to describe complex splicing events. For instance, the figure to the right shows 3 spliceforms from the mouse hyaluronidase
Hyaluronidase
The hyaluronidases are a family of enzymes that degrade hyaluronic acid.In humans, there are six associated genes, including HYAL1, HYAL2, HYAL3, and PH-20/SPAM1.-Use as a drug:...
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.
General splicing mechanism
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
, it includes several intron
Intron
An intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of 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...
s and exon
Exon
An exon is a nucleic acid sequence that is represented in the mature form of an RNA molecule either after portions of a precursor RNA have been removed by cis-splicing or when two or more precursor RNA molecules have been ligated by trans-splicing. The mature RNA molecule can be a messenger RNA...
s. (In nematode
Nematode
The nematodes or roundworms are the most diverse phylum of pseudocoelomates, and one of the most diverse of all animals. Nematode species are very difficult to distinguish; over 28,000 have been described, of which over 16,000 are parasitic. It has been estimated that the total number of nematode...
s, the mean is 4–5 exons and introns; in the fruit fly Drosophila
Drosophila melanogaster
Drosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...
there can be more than 100 introns and exons in one transcribed pre-mRNA.) 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. In humans the branch consensus is yUnAy. The branch site is followed by a series of pyrimidine
Pyrimidine
Pyrimidine is a heterocyclic aromatic organic compound similar to benzene and pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring...
s, or polypyrimidine tract, then by AG at 3' end.
Splicing of mRNA is performed by an RNA and protein complex known as the spliceosome
Spliceosome
A spliceosome is a complex of snRNA and protein subunits that removes introns from a transcribed pre-mRNA segment. This process is generally referred to as splicing.-Composition:...
, containing snRNP
SnRNP
snRNPs , or small nuclear ribonucleoproteins, are RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs...
s designated U1, U2, U4, U5, and U6 (U3 is not involved in mRNA splicing). U1 binds to 5' GU and U2 binds to branch site (A) with the assistance of the U2AF
U2AF2
Splicing factor U2AF 65 kDa subunit is a protein that in humans is encoded by the U2AF2 gene.-Interactions:U2AF2 has been shown to interact with WT1, PUF60, SFRS2IP, SRPK2, SF1, U2 small nuclear RNA auxiliary factor 1 and SFRS11.-References:...
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.
The U4,U5,U6 complex binds, and U6 replaces the U1 position. U1 and U4 leave. The remaining complex then performs two transesterification
Transesterification
In organic chemistry, transesterification is the process of exchanging the organic group R″ of an ester with the organic group R′ of an alcohol. These reactions are often catalyzed by the addition of an acid or base catalyst...
reactions. In the first transesterification, 5' end of the intron is cleaved from the upstream exon and joined to the branch site A by a 2',5'-phosphodiester linkage. In the second transesterification, the 3' end of the intron is cleaved from the downstream exon, and the two exons are joined by a phosphodiester bond. The intron is then released in lariat form and degraded.
Regulatory elements and proteins
Trans-acting
In the field of molecular biology, trans-acting , in general, means "acting from a different molecule"...
proteins (repressors and activators), corresponding cis-acting regulatory sites (silencers and enhancers) on the RNA, and other RNA features that influence how splicing will occur, such as secondary structures. Together, these elements form a "splicing code" that governs how splicing will occur under different cellular conditions
.
The determinants of splicing act in an inter-dependent fashion, but in many cases a particular determinant will have a consistent influence on splicing. Among these cases, there are two major types of cis-acting RNA sequence elements present in pre-mRNAs and they have corresponding trans-acting
Trans-acting
In the field of molecular biology, trans-acting , in general, means "acting from a different molecule"...
RNA-binding protein
RNA-binding protein
RNA-binding proteins are proteins that bind to RNA. They bind to either double-strand or single-strand RNAs through RNA recognition motif . RNA-binding proteins may regulate the translation of RNA, and post-transcriptional events, such as RNA splicing, editing.They are cytoplasmic and nuclear...
s. Splicing silencers are sites to which splicing repressor proteins bind, reducing the probability that a nearby site will be used as a splice junction. These can be located in the intron itself (intronic splicing silencers, ISS) or in a neighboring exon (exonic splicing silencer
Exonic splicing silencer
An exonic splicing silencer is a small region of an exon that inhibits or silences splicing of the pre-mRNA....
s, ESS). They vary in sequence, as well as in the types of proteins that bind to them. The majority of splicing repressors are heterogeneous nuclear ribonucleoproteins (hnRNPs) such as hnRNPA1 and polypyrimidine tract binding protein (PTB).
Exonic splicing enhancer
An exonic splicing enhancer is a DNA sequence motif consisting of 6 bases within an exon that directs, or enhances, accurate splicing of hetero-nuclear RNA or pre-mRNA into messenger RNA .- The Basics :...
s, ESE). Most of the activator proteins that bind to ISEs and ESEs are members of the SR protein
SR protein
SR proteins are serine/arginine-rich proteins which are involved in regulating and selecting splice sites in eukaryotic mRNA. Alternative splicing requires SR proteins, which select the alternative splice sites to be utilized...
family. Such proteins contain RNA recognition motifs and arginine and serine-rich (RS) domains.
The adaptive significance of splicing silencers and enhancers is attested by a study showing that there is strong selection in human genes against mutations that produce new silencers or disrupt existing enhancers.
In general, the determinants of splicing work in an inter-dependent manner that depends on context, so that the rules governing how splicing is regulated form a splicing code. The presence of a particular cis-acting RNA sequence element may increase the probability that a nearby site will be spliced in some cases, but decrease the probability in other cases, depending on context. The context within which regulatory elements act includes cis-acting context that is established by the presence of other RNA sequence features, and trans-acting
Trans-acting
In the field of molecular biology, trans-acting , in general, means "acting from a different molecule"...
context that is established by cellular conditions. For example, some cis-acting RNA sequence elements influence splicing only if multiple elements are present in the same region so as to establish context. As another example, a cis-acting element can have opposite effects on splicing, depending on which proteins are expressed in the cell (e.g., neuronal versus non-neuronal PTB).
Exon skipping: Drosophila dsx
Doublesex
Doublesex is a gene that is involved in the sex determination system of the fruit fly Drosophila melanogaster.-Sex determination:...
contain 6 exons. In males, exons 1,2,3,5,and 6 are joined to form the mRNA, which encodes a transcriptional regulatory protein required for male development. In females, exons 1,2,3, and 4 are joined, and a polyadenylation
Polyadenylation
Polyadenylation is the addition of a poly tail to an RNA molecule. The poly tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA for translation...
signal in exon 4 causes cleavage of the mRNA at that point. The resulting mRNA is a transcriptional regulatory protein required for female development.
This is an example of exon skipping. The intron upstream from exon 4 has a polypyrimidine tract that doesn't match the consensus sequence well, so that U2AF proteins bind poorly to it without assistance from splicing activators. This 3' splice acceptor site is therefore not used in males. Females, however, produce the splicing activator Transformer (Tra)(see below). The SR protein Tra2 is produced in both sexes and binds to an ESE in exon 4; if Tra is present, it binds to Tra2 and, along with another SR protein, forms a complex that assists U2AF proteins in binding to the weak polypyrimidine tract. U2 is recruited to the associated branch point, and this leads to inclusion of exon 4 in the mRNA.
Alternative acceptor sites: Drosophila Transformer
Drosophila melanogaster
Drosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...
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
Stop codon
In the genetic code, a stop codon is a nucleotide triplet within messenger RNA that signals a termination of translation. Proteins are based on polypeptides, which are unique sequences of amino acids. Most codons in messenger RNA correspond to the addition of an amino acid to a growing polypeptide...
. The resulting mRNA encodes a truncated protein product that is inactive. Females produce the master sex determination protein Sex lethal (Sxl). The Sxl protein is a splicing repressor that binds to an ISS in the RNA of the Tra transcript near the upstream acceptor site, preventing U2AF
U2AF2
Splicing factor U2AF 65 kDa subunit is a protein that in humans is encoded by the U2AF2 gene.-Interactions:U2AF2 has been shown to interact with WT1, PUF60, SFRS2IP, SRPK2, SF1, U2 small nuclear RNA auxiliary factor 1 and SFRS11.-References:...
protein from binding to the polypyrimidine tract. This prevents the use of this junction, shifting the spliceosome binding to the downstream acceptor site. Splicing at this point bypasses the stop codon, which is excised as part of the intron. The resulting mRNA encodes an active Tra protein, which itself is a regulator of alternative splicing of other sex-related genes (see dsx above).
Exon definition: Fas receptor
Fas receptor
The FAS receptor also known as apoptosis antigen 1 , cluster of differentiation 95 or tumor necrosis factor receptor superfamily member 6 is a protein that in humans is encoded by the TNFRSF6 gene....
protein are produced by alternative splicing. Two normally occurring isoforms in humans are produced by an exon-skipping mechanism. An mRNA including exon 6 encodes the membrane-bound form of the Fas receptor, which promotes apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
, 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
TIA1
TIA1 is a poly-binding protein. It is associated with programmed cell death and regulates alternative splicing of the gene encoding the Fas receptor, an apoptosis-promoting protein....
and polypyrimidine tract-binding protein (PTB).
- The 5' donor site in the intron downstream from exon 6 in the pre-mRNA has a weak agreement with the consensus sequence, and is not bound usually by the U1 snRNP. If U1 does not bind, the exon is skipped (see "a" in accompanying figure).
- 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 (see b).
- 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 (see c).
This mechanism is an example of exon definition in splicing. A spliceosome assembles on an intron, and the snRNP subunits bring fold the RNA so that the 5' and 3' ends of the intron are joined. However, recently studied examples such as this one show that there are also interactions between the ends of the exon. In this particular case, these exon definition interactions are necessary to allow the binding of core splicing factors prior to assembly of the spliceosomes on the two flanking introns.
Repressor-activator competition: HIV-1 tat exon 2
HIV
Human immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive...
, the retrovirus
Retrovirus
A retrovirus is an RNA virus that is duplicated in a host cell using the reverse transcriptase enzyme to produce DNA from its RNA genome. The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA...
that causes AIDS
AIDS
Acquired immune deficiency syndrome or acquired immunodeficiency syndrome is a disease of the human immune system caused by the human immunodeficiency virus...
in humans, produces a single primary RNA transcript, which is alternatively spliced in multiple ways to produce over 40 different mRNAs. Equilibrium among differentially spliced transcripts provides multiple mRNAs encoding different products that are required for viral multiplication. One of the differentially spliced transcripts contains the tat gene, in which exon 2 is a cassette exon that may be skipped or included. The inclusion of tat exon 2 in the RNA is regulated by competition between the splicing repressor hnRNP A1 and the SR protein SC35. Within exon 2 an exonic splicing silencer sequence (ESS) and an exonic splicing enhancer sequence (ESE) overlap. If A1 repressor protein binds to the ESS, it initiates cooperative binding of multiple A1 molecules, extending into the 5’ donor site upstream of exon 2 and preventing the binding of the core splicing factor U2AF35 to the polypyrimidine tract. If SC35 binds to the ESE, it prevents A1 binding and maintains the 5’ donor site in an accessible state for assembly of the spliceosome. Competition between the activator and repressor ensures that both mRNA types (with and without exon 2) are produced.
Adaptive significance
Alternative splicing is one of several exceptions to the original idea that one DNA sequence codes for one polypeptide (the One gene-one enzyme hypothesisOne gene-one enzyme hypothesis
The one gene-one enzyme hypothesis is the idea that genes act through the production of enzymes, with each gene responsible for producing a single enzyme that in turn affects a single step in a metabolic pathway...
). 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. Since the methods of regulation are inherited, this provides novel ways for mutations to affect gene expression.
It has been proposed that for eukaryote
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
s alternative splicing was a very important step towards higher efficiency, because information can be stored much more economically. Several proteins can be encoded by a single gene, rather than requiring a separate gene for each, and thus allowing a more varied proteome
Proteome
The proteome is the entire set of proteins expressed by a genome, cell, tissue or organism. More specifically, it is the set of expressed proteins in a given type of cells or an organism at a given time under defined conditions. The term is a portmanteau of proteins and genome.The term has been...
from a genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
of limited size. 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
Protein isoform
A protein isoform is any of several different forms of the same protein. Different forms of a protein may be produced from related genes, or may arise from the same gene by alternative splicing. A large number of isoforms are caused by single-nucleotide polymorphisms or SNPs, small genetic...
without loss of the original protein. Comparative studies indicate that alternative splicing preceded multicellularity in evolution, and suggest that this mechanism might have been co-opted to assist in the development of multicellular organisms.
Research based on the Human Genome Project
Human Genome Project
The Human Genome Project is an international scientific research project with a primary goal of determining the sequence of chemical base pairs which make up DNA, and of identifying and mapping the approximately 20,000–25,000 genes of the human genome from both a physical and functional...
and other genome sequencing has shown that humans have only about 30% more genes than the roundworm Caenorhabditis elegans
Caenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
, and only about twice as many as the fly Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...
. 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 EST
Expressed sequence tag
An expressed sequence tag or EST is a short sub-sequence of a cDNA sequence. They may be used to identify gene transcripts, and are instrumental in gene discovery and gene sequence determination. The identification of ESTs has proceeded rapidly, with approximately 65.9 million ESTs now available in...
s each from human, mouse, rat, cow, fly (D. melanogaster), worm (C. elegans), and the plant Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana is a small flowering plant native to Europe, Asia, and northwestern Africa. A spring annual with a relatively short life cycle, arabidopsis is popular as a model organism in plant biology and genetics...
found no large differences in frequency of alternatively spliced genes among humans and any of the other animals tested. Another study, however, proposed that these results were an artifact of the different numbers of ESTs available for the various organisms. When they compared alternative splicing frequencies in random subsets of genes from each organism, the authors concluded that vertebrates do have higher rates of alternative splicing than invertebrates.
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 mutationMutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
s 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
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
of trans-acting splicing factors. Others may be produced by changes in the relative amounts of splicing factors produced; for instance, breast cancer cells have been shown to have increased levels of the splicing factor SF2/ASF. One study found that a relatively small percentage (383 out of over 26000) 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. It is believed however that the deleterious effects of mis-spliced transcripts are usually safeguarded and eliminated by a cellular posttranscriptional quality control mechanism termed Nonsense-mediated mRNA decay [NMD].
One example of a specific splicing variant associated with cancers is in one of the human DNMT genes. Three DNMT genes encode enzymes that add methyl
DNA methylation
DNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring...
groups to DNA, a modification that often has regulatory effects. Several abnormally spliced DNMT3B mRNAs are found in tumors and cancer cell lines. In two separate studies, expression of two of these abnormally spliced mRNAs in mammalian cells caused changes in the DNA methylation patterns in those cells. Cells with one of the abnormal mRNAs also grew twice as fast as control cells, indicating a direct contribution to tumor development by this product.
Another example is the Ron (MST1R
MST1R
Macrophage-stimulating protein receptor is a protein that in humans is encoded by the MST1R gene.-Further reading:...
) proto-oncogene. An important property of cancerous cells is their ability to move and invade normal tissue. Production of an abnormally spliced transcript of Ron has been found to be associated with increased levels of the SF2/ASF in breast cancer cells. The abnomal isoform of the Ron protein encoded by this mRNA leads to cell motility.
Recent provocative studies point to a key function of chromatin structure and histone modifications in alternative splicing regulation. These insights suggest that epigenetic regulation determines not only what parts of the genome are expressed but also how they are spliced.
Genome-wide analysis of alternative splicing
Genome-wide analysis of alternative splicing is a challenging task. Typically, alternatively spliced transcripts have been found by comparing ESTExpressed sequence tag
An expressed sequence tag or EST is a short sub-sequence of a cDNA sequence. They may be used to identify gene transcripts, and are instrumental in gene discovery and gene sequence determination. The identification of ESTs has proceeded rapidly, with approximately 65.9 million ESTs now available in...
sequences, but this requires sequencing of very large numbers of ESTs. Most EST libraries come from a very limited number of tissues, so tissue-specific splice variants are likely to be missed in any case. High-throughput approaches to investigate splicing fall into three categories; DNA microarray
DNA microarray
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
-based analyses, CLIP, and in vivo reporter gene
Reporter gene
In molecular biology, a reporter gene is a gene that researchers attach to a regulatory sequence of another gene of interest in cell culture, animals or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and...
assays.
In microarray analysis, arrays of DNA fragments representing individual exon
Exon
An exon is a nucleic acid sequence that is represented in the mature form of an RNA molecule either after portions of a precursor RNA have been removed by cis-splicing or when two or more precursor RNA molecules have been ligated by trans-splicing. The mature RNA molecule can be a messenger RNA...
s (e.g. Affymetrix
Affymetrix
Affymetrix is a company that manufactures DNA microarrays; it is based in Santa Clara, California, United States. The company was founded by Dr. Stephen Fodor in 1992. It began as a unit in Affymax N.V...
exon microarray) or exon/exon boundaries (e.g. arrays from ExonHit or Jivan) have been used. The array is then probed with labeled cDNA from tissues of interest. The probe cDNAs bind to DNA from the exons that are included in mRNAs in their tissue of origin, or to DNA from the boundary where two exons have been joined. This can reveal the presence of particular alternatively spliced mRNAs. Deep sequencing
RNA-Seq
RNA-seq, also called "Whole Transcriptome Shotgun Sequencing" and dubbed "a revolutionary tool for transcriptomics", refers to the use of high-throughput sequencing technologies to sequence cDNA in order to get information about a sample's RNA content, a technique that is quickly becoming...
technologies are also being used to perform genome-wide studies of transcript variation.
CLIP (Cross-linking and immunoprecipitation
Immunoprecipitation
Immunoprecipitation is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins...
) uses UV radiation to link proteins to RNA molecules in a tissue during splicing. A trans-acting splicing regulatory protein of interest is then precipitated using specific antibodies. When the RNA attached to that protein is isolated and cloned, it reveals the target sequences for that protein.
Finally, it is possible to find the splicing proteins involved in a specific alternative splicing event by constructing reporter genes that will express one of two different fluorescent proteins depending on the splicing reaction that occurs. This method has been used to isolate mutants affecting splicing and thus to identify novel splicing regulatory proteins inactivated in those mutants.
External links
- A General Definition and Nomenclature for Alternative Splicing Events at SciVeeSciVeeSciVee is a science video sharing website where researchers can upload, view and share science video clips and connect them to scientific literature, posters and slides...
- AStalavista (Alternative Splicing landscape visualization tool), a method for the computationally exhaustive classification of Alternative Splicing Structures
- Stamms-lab.net: Research Group dealing with alternative Splicing issues and mis-splicing in human diseases
- Alternative Splicing of ion channels in the brain, connected to mental and neurological diseases
- BIPASS: Web Services in Alternative Splicing
See also
- AspicDBAspicDBASPicDB is a database of human protein variants generated by alternative splicing, a process by which the exons of the RNA produced by transcription of a gene are reconnected in multiple ways during RNA splicing....
database - Alternative polyadenylation (section of Polyadenylation)