Figure 1. Alternative splicing generates transcriptome diversity and enables gene regulation. It can generate mRNAs that encode proteins with different or even opposite functions. Figure used with permission. Well, it plays a crucial role in generating biological complexity and proteomic diversity in humans and significantly affects various functions in cellular processes, tissue specificity, developmental states, and disease conditions.
As a result, alternative splicing is often involved in human disease e. In molecular biology a cis-acting regulatory element is defined as a region of chromosomal DNA that regulates the transcription or expression of a gene that is found on the same chromosome.
A trans-acting regulatory element, on the other hand, is a protein that binds to a cis-acting element of a specific gene to regulate its transcription. As a result, cis-acting regulatory elements in pre-mRNA sequences determine which exons are retained and which exons are spliced out. These elements alter splicing by binding different trans-acting protein factors such as serine—arginine-rich SR proteins, which function as splicing facilitators, and heterogeneous nuclear ribonucleoproteins hnRNPs , which suppress splicing.
Silencing can be inhibited sterically when splicing inhibitors bind to splicing silencers that are found near splicing enhancers. This prevents the binding of small nuclear ribonucleoproteins snRNPs and other activator proteins or prevents spliceosome assembly.
The inclusion or splicing of an alternative exon is therefore determined by combinatorial effects, cellular abundance, and competitive binding between SR activators and hnRNP inhibitors. Alternative splicing outcomes depend on the stoichiometry and interactions of splicing activators and inhibitors, as well as the steric conformation and accessibility of the splicing sites. On average, human transcripts contain approximately nine introns. Advances in high-throughput technologies, including next-generation RNA and DNA sequencing, have facilitated studies of genome-wide alternative splicing.
Alternative splicing events are differentially regulated across different tissues and during development, as well as among individuals and populations. Studies indicate that alternative splicing of CD44, a protein involved in T-cell homing with 10 variable cassette exons and six distinct protein isoforms, is crucial for T-cell function. The variable exons of CD44 encode portions of the membrane-proximal extracellular domain of the protein, and the presence of some of the variable exons has been shown to increase the association of CD44 with various proteins.
RNA splicing was initially discovered in the s, overturning years of thought in the field of gene expression. Gene regulation was first studied most thoroughly in relatively simple bacterial systems. Most bacterial RNA transcripts do not undergo splicing; these transcripts are said to be colinear, with DNA directly encoding them.
However, in , several groups of researchers who were working with adenoviruses that infect and replicate in mammalian cells obtained some surprising results.
These scientists identified a series of RNA molecules that they termed "mosaics," each of which contained sequences from noncontiguous sites in the viral genome Berget et al. These mosaics were found late in viral infection. Studies of early infection revealed long primary RNA transcripts that contained all of the sequences from the late RNAs, as well as what came to be called the intervening sequences introns.
Subsequent to the adenoviral discovery, introns were found in many other viral and eukaryotic genes, including those for hemoglobin and immunoglobulin Darnell, Splicing of RNA transcripts was then observed in several in vitro systems derived from eukaryotic cells, including removal of introns from transfer RNA in yeast cell-free extracts Knapp et al.
These observations solidified the hypothesis that splicing of large initial transcripts did, in fact, yield the mature mRNA. Other hypotheses proposed that the DNA template in some way looped or assumed a secondary structure that allowed transcription from noncontiguous regions Darnell, Splicing occurs in several steps and is catalyzed by small nuclear ribonucleoproteins snRNPs , commonly pronounced "snurps".
The bonding of the guanine and adenine bases takes place via a chemical reaction known as transesterification , in which a hydroxyl OH group on a carbon atom of the adenine "attacks" the bond of the guanine nucleotide at the splice site. The guanine residue is thus cleaved from the RNA strand and forms a new bond with the adenine. The adjoining exons are covalently bound, and the resulting lariat is released with U2, U5, and U6 bound to it. In addition to consensus sequences at their splice sites, eukaryotic genes with long introns also contain exonic splicing enhancers ESEs.
These sequences, which help position the splicing apparatus, are found in the exons of genes and bind proteins that help recruit splicing machinery to the correct site. Most splicing occurs between exons on a single RNA transcript, but occasionally trans-splicing occurs, in which exons on different pre-mRNAs are ligated together.
The splicing process occurs in cellular machines called spliceosomes, in which the snRNPs are found along with additional proteins. The primary variety of spliceosome is one of the most plentiful structures in the cell, and recently, a secondary type of spliceosome has been identified that processes a minor category of introns.
These introns are referred to as Utype introns because they depend upon the action of a snRNP called U12 the common introns described above are called U2-type introns.
Some RNA molecules have the capacity to splice themselves; the initial discovery of this self-splicing ability in the protozoan Tetrahymena thermophila was recognized with the Nobel Prize in The self-splicing introns found in T. Group I introns all fold into a complex secondary structure with nine loops and employ transesterification reactions as described above.
On the other hand, Group II self-splicing introns are found in mitochondrial genes and are excised by a mechanism that bears similarities to pre-mRNA splicing, including the production of lariats. For this reason, it has been proposed that perhaps pre-mRNA introns and splicing mechanisms evolved from the Group II introns. Early in the course of splicing research, yet another surprising discovery was made; specifically, researchers noticed that not only was pre-mRNA punctuated by introns that needed to be excised, but also that alternative patterns of splicing within a single pre-mRNA molecule could yield different functional mRNAs Figure 2; Berget et al.
The first example of alternative splicing was defined in the adenovirus in and demonstrated that one pre-mRNA molecule could be spliced at different junctions to result in a variety of mature mRNA molecules, each containing different combinations of exons.
Shortly afterward, alternative splicing was found to occur in cellular genes as well, with the first example identified in the IgM gene, a member of the immunoglobulin superfamily Early et al.
Another example of a gene with an impressive number of alternative splicing patterns is the Dscam gene from Drosophila , which is involved in guiding embryonic nerves to their targets during formation of the fly's nervous system. Examination of the Dscam sequence reveals such a large number of introns that differential splicing could, in theory, create a staggering 38, different mRNAs. This ability to create so many mRNAs may provide the diversity necessary for forming a complex structure such as the nervous system Schmucker et al.
In fact, the existence of multiple mRNA transcripts within single genes may account for the complexity of some organisms, such as humans, that have relatively few genes approximately 20, For example, work from Wang et al. The existence of introns and differential splicing helps explain how new genes are created during evolution. Splicing makes genes more "modular," allowing new combinations of exons to be created during evolution.
Furthermore, new exons can be inserted into old introns, creating new proteins without disrupting the function of the old gene. During a typical gene splicing event, the pre-mRNA transcribed from one gene can lead to different mature mRNA molecules that generate multiple functional proteins.
Thus, gene splicing enables a single gene to increase its coding capacity, allowing the synthesis of protein isoforms that are structurally and functionally distinct. Gene splicing is observed in high proportion of genes.
There are several types of common gene splicing events. These are the events that can simultaneously occur in the genes after the mRNA is formed from the transcription step of the central dogma of molecular biology. Exon Skipping: This is the most common known gene splicing mechanism in which exon s are included or excluded from the final gene transcript leading to extended or shortened mRNA variants. The exons are the coding regions of a gene and are responsible for producing proteins that are utilized in various cell types for a number of functions.
Follow-up work revealed that the expression levels of CELF and MBNL are inversely tied to one another during muscle development, and that they antagonistically regulate more than 1, pre-mRNA transcripts, some of which are translated into proteins critical for muscle contraction.
Since the early efforts to describe splicing, the textbook view of the process has been that it occurs post-transcriptionally.
Karla Neugebauer and her lab at Yale University champion this model and use biochemical and computational approaches to study the phenomenon. Last year, an international team of researchers published on the in vivo consequences of such co-transcriptional splicing, showing that mouse embryonic stem cells with a knocked-in gene for a slow-transcribing version of RNAPII exhibit neuronal differentiation defects due to the failure to properly splice genes involved in synapse signaling.
Researchers are also exploring the possibility that chromatin architecture and epigenetics serve as another layer of splicing regulation by modulating the rate of RNAPII transcription. Despite a collection of cases teasing apart the mechanism of alternative splicing and highlighting its functional consequences, the number of uncharacterized splicing events is immense, and the pages documenting the physiological importance of alternative splicing largely remain blank.
Titin , which codes for a protein in muscle, is one example of a gene whose pre-mRNA transcript can be spliced in multiple ways to yield different protein isoforms.
During development of the fetal heart, more exons are left in during splicing, which produces a relatively long, springy protein. The result is a relatively short, stiff protein. If RBM20 is missing or defective in adult hearts, these hearts will produce more fetal, springy titin protein relative to the stiff adult version. This is thought to reduce the capacity of the heart to contract, contributing to a condition known as dilated cardiomyopathy.
More than one-third of disease-causing mutations map to sites bound by the spliceosome or RBPs, or to RBP-encoding gene regions. Therefore, mis-splicing has a strong potential to be implicated in disease. One scenario involves the titin TTN gene, which holds the record for the highest number of exons—a whopping —among all mammalian genes and encodes the largest known protein in the human body, weighing in at 4.
The TTN protein is a molecular spring that contributes to the elasticity of heart muscle. Over the course of cardiac development, there is a gradual increase in the frequency of TTN exon skipping by the spliceosome, and these exons are thus spliced out from the mRNA. See illustration on opposite page. The transition from the fetal to the adult cardiac titin isoform is part of the normal developmental program and is orchestrated by an RBP called RBM Using a rodent model, an international cohort of researchers and physicians demonstrated that the absence of RBM20 causes TTN mis-splicing, leading to the buildup of long, elastic TTN and phenotypes resembling the decreased heart contractility seen in humans with dilated cardiomyopathy induced by mutations in RBM Another example is DMD , the gene that encodes the dystrophin protein, which is important for muscle integrity and force transmission.
Mutational variants in DMD are notoriously associated with Duchenne muscular dystrophy, a disease that severely impairs muscle function. One disease-causing DMD mutation is a multiexon deletion that commonly results in a frameshift starting at exon Splicing the remaining exons together results in a shortened dystrophin protein with compromised function.
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