Alternative splicing

Alternative splicing was first observed in 1977. The adenovirus produces five primary transcripts early in its infectious cycle, prior to viral DNA replication, and an additional one later, after DNA replication begins. The early primary transcripts continue to be produced after DNA replication begins. The additional primary transcript produced late in infection is large and comes from 5/6 of the 32kb adenovirus genome. This is much larger than any of the individual adenovirus mRNAs present in infected cells. Researchers found that the primary RNA transcript produced by adenovirus type 2 in the late phase was spliced in many different ways, resulting in mRNAs encoding different viral proteins. In addition, the primary transcript contained multiple polyadenylation 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. Examples of alternative splicing in immunoglobin gene transcripts in mammals were also observed in the early 1980s. Since then, many other examples of biologically relevant alternative splicing have been found in eukaryotes. In 2021, it was discovered that the genome of adenovirus type 2, the adenovirus in which alternative splicing was first identified, was able to produce a much greater variety of splice variants than previously thought. By using next generation sequencing technology, researchers were able to update the human adenovirus type 2 transcriptome and document the presence of 904 splice variants produced by the virus through a complex pattern of alternative splicing. Very few of these splice variants have been shown to be functional, a point that the authors raise in their paper. ::"An outstanding question is what roles the menagerie of novel RNAs play or whether they are spurious molecules generated by an overloaded splicing machinery." ==Modes==

may be spliced out of the primary transcript or retained. This is the most common mode in mammalian pre-mRNAs. 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 for the transcript. Both of these mechanisms are found in combination with alternative splicing and provide additional variety in mRNAs derived from a gene. gene. Directionality of transcription from 5' to 3' is shown from left to right. Exons and introns are not drawn to scale. These modes describe basic splicing mechanisms, but may be inadequate to describe complex splicing events. For instance, the figure to the right shows three 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. ==Mechanisms==

General splicing mechanism s – the polypyrimidine tract – then by AG at the 3' end. U1 binds to the 5' GU and U2, with the assistance of the U2AF protein factors, binds to the branchpoint A within the branch site. 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. Regulatory elements and proteins Splicing is regulated by trans-acting proteins (repressors and activators) and corresponding cis-acting regulatory sites (silencers and enhancers) on the pre-mRNA. However, as part of the complexity of alternative splicing, it is noted that the effects of a splicing factor are frequently position-dependent. That is, a splicing factor that serves as a splicing activator when bound to an intronic enhancer element may serve as a repressor when bound to its splicing element in the context of an exon, and vice versa. The secondary structure of the pre-mRNA transcript also plays a role in regulating splicing, such as by bringing together splicing elements or by masking a sequence that would otherwise serve as a binding element for a splicing factor. Together, these elements form a "splicing code" that governs how splicing will occur under different cellular conditions. There are two major types of cis-acting RNA sequence elements present in pre-mRNAs and they have corresponding trans-acting RNA-binding proteins. 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 silencers, 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). Examples Exon skipping: Drosophila dsx Pre-mRNAs from the Drosophila melanogaster gene dsx 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 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 does not 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 branchpoint, and this leads to inclusion of exon 4 in the mRNA. Alternative acceptor sites: Drosophila '''' Pre-mRNAs of the Transformer (Tra) gene of Drosophila melanogaster 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. 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 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). 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 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. • 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 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. 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==

Genuine alternative splicing occurs in both protein-coding genes and non-coding genes to produce multiple products (proteins or non-coding RNAs). External information is needed in order to decide which product is made, given a DNA sequence and the initial transcript. Since the methods of regulation are inherited, this provides novel ways for mutations to affect gene expression. suggesting that protein isoforms may display functional diversity due to the alteration of functional modules within these regions. Such functional diversity achieved by isoforms is reflected by their expression patterns and can be predicted by machine learning approaches. 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 and other genome sequencing has shown that humans have only about 30% more genes than the roundworm Caenorhabditis elegans, and only about twice as many as the fly Drosophila melanogaster. 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 expressed sequence tags (EST) each from human, mouse, rat, cow, fly (D. melanogaster), worm (C. elegans), and the plant Arabidopsis thaliana 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. ==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 study in 2005 involving probabilistic analyses indicated that greater than 60% of human disease-causing mutations affect splicing rather than directly affecting coding sequences. A more recent study indicates that one-third of all hereditary diseases are likely to have a splicing component. As described below, a prominent example of splicing-related diseases is cancer. Abnormally spliced mRNAs are also found in a high proportion of cancerous cells. Combined RNA-Seq and proteomics analyses have revealed striking differential expression of splice isoforms of key proteins in important cancer pathways. It is not always clear whether such aberrant patterns of splicing contribute to the cancerous growth, or are merely consequence of cellular abnormalities associated with cancer. For certain types of cancer, like in colorectal and prostate, the number of splicing errors per cancer has been shown to vary greatly between individual cancers, a phenomenon referred to as transcriptome instability. Transcriptome instability has further been shown to correlate greatly with reduced expression level of splicing factor genes. Mutation of DNMT3A has been demonstrated to contribute to hematologic malignancies, and that DNMT3A-mutated cell lines exhibit transcriptome instability as compared to their isogenic wildtype counterparts. In fact, 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 be due to the high frequency of somatic mutations in splicing factor genes, 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 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. 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-scale (transcriptome-wide) analysis==

Transcriptome-wide analysis of alternative splicing is typically performed by high-throughput RNA-sequencing. Most commonly, by short-read sequencing, such as by Illumina instrumentation. But even more informative, by long-read sequencing, such as by Nanopore or PacBio instrumentation. Transcriptome-wide analyses can for example be used to measure the amount of deviating alternative splicing, such as in a cancer cohort. Deep sequencing technologies have been used to conduct genome-wide analyses of both unprocessed and processed mRNAs; thus providing insights into alternative splicing. For example, results from use of deep sequencing indicate that, in humans, an estimated 95% of transcripts from multiexon genes undergo alternative splicing, with a number of pre-mRNA transcripts spliced in a tissue-specific manner. More historically, alternatively spliced transcripts have been found by comparing EST 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 have, however, been developed, such as: DNA microarray–based analyses, RNA-binding assays, and deep sequencing. These methods can be used to screen for polymorphisms or mutations in or around splicing elements that affect protein binding. When combined with splicing assays, including in vivo reporter gene assays, the functional effects of polymorphisms or mutations on the splicing of pre-mRNA transcripts can then be analyzed. In microarray analysis, arrays of DNA fragments representing individual exons (e.g. Affymetrix 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. CLIP (Cross-linking and immunoprecipitation) 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. This method involves an adaptation of the "Systematic Evolution of Ligands by Exponential Enrichment (SELEX)" method together with a microarray-based readout. Use of the MEGAshift method has provided insights into the regulation of alternative splicing by allowing for the identification of sequences in pre-mRNA transcripts surrounding alternatively spliced exons that mediate binding to different splicing factors, such as ASF/SF2 and PTB. This approach has also been used to aid in determining the relationship between RNA secondary structure and the binding of splicing factors. Recent advancements in protein structure prediction have facilitated the development of new tools for genome annotation and alternative splicing analysis. For instance, isoform.io, a platform guided by protein structure predictions, has evaluated hundreds of thousands of isoforms of human protein-coding genes assembled from numerous RNA sequencing experiments across a variety of human tissues. This comprehensive analysis has led to the identification of numerous isoforms with more confidently predicted structure and potentially superior function compared to canonical isoforms in the latest human gene database. By integrating structural predictions with expression and evolutionary evidence, this approach has demonstrated the potential of protein structure prediction as a tool for refining the annotation of the human genome. ==Databases==

There is a collection of alternative splicing databases. These databases are useful for finding genes having pre-mRNAs undergoing alternative splicing and alternative splicing events or to study the functional impact of alternative splicing. • AspicDB database • Intronerator database • ProSAS database == See also ==