Alternative pre‐mRNA splicing

Have you ever felt ashamed of yourself when using copy and paste instead of writing an original? You do not have to! Nature is using copy and paste with incredibly great success, and we can learn a lot by studying this intriguing method called alternative splicing. If you compare the mRNA with genomic sequences, you will notice that they are quite different. Just before the pre-mRNA is exported to the cytosol, the introns are excised by pre-mRNA splicing and remain in the nucleus. So, only a small proportion of the primary transcript—the exons—are linked together, forming the mature mRNA. The key player of pre-mRNA splicing is the macromolecular complex called spliceosome. It is composed of 5 small nuclear RNAs (snRNAs) and more than 150 additional proteins. The spliceosome recognizes exon-intron junctions and removes introns. Exons are defined by three major sequence elements: the 5′ splice site, the 3′ splice site and the branch point (Figure 1A). The components of the spliceosome recognize these elements and assemble in a stepwise manner on the nascent pre-mRNA. Initially, the U1 snRNP complex, formed by U1 snRNA and proteins, binds to the 5′ splice site, and then, U2 snRNP or splicing factor 1 (SF1) binds to the branch point sequence, which facilitates binding of the U2AF factor on the 3′ splice site. Because these splice sites only follow loose consensus sequences, additional factors called hnRNPs and SR proteins are required for exon recognition. Due to protein-RNA and protein-protein interactions with components of the spliceosome, these proteins usually stabilize the binding of U1 and U2 to form the prespliceosomal A complex. Through exchange and recruitment of additional factors, the A complex is transformed into the spliceosomal B complex that removes the intron and joins the exons.1 Many organisms take advantage of the opportunity to insert or remove single exons—either completely or in parts—from the mRNA. This process is called alternative splicing. Given the surprisingly low number of human genes, alternative splicing represents an elegant mechanism to generate a high diversity of proteins with various functions from a single pre-mRNA molecule.2 Alternative splicing is enabled by two major groups of proteins, hnRNPs and SR proteins, which bind to splicing regulatory RNA elements (enhancers or silencers) that can either be exonic or intronic (Figure 1A). As a general rule, SR proteins mostly bind to enhancers promoting exon inclusion, while hnRNPs preferentially bind to silencers antagonizing exon inclusion. SR proteins and hnRNPs generally bind weakly to RNA. An increased specificity is achieved by the combination of multiple weak interactions between the main splice machinery and additional regulatory proteins and signals on the pre-mRNA. The formation of a specific protein-RNA complex by combining several weak interactions has the advantage of being highly flexible, allowing a dynamic interaction with mRNA binding sites due to the possible exchange of spliceosomal components. Therefore, alternative splicing depends on the cell type, developmental stage and environmental influences.3 Beside this general concept, other mechanisms of alternative pre-mRNA splicing have emerged. The protein kinase and endoribonuclease IRE1 have been shown to bind and cleave specific mRNAs in response to endoplasmic reticulum (ER) stress. Upon accumulation of unfolded proteins in the ER, the XBP1 mRNA is processed to an active form by the unconventional splicing mechanism utilizing IRE1.4 The resulting loss of 26 nucleotides causes an isoform sXBP1 with increased transcription factor activity. Therefore, the expression levels of sXBP1 have been used as a marker for ER stress.5, 6 Alternative pre-mRNA splicing emerges as a central element of gene regulation that affects binding properties, intracellular localization, enzymatic activity, stability and post-translational modification of a large number of proteins and interferes with almost every biological function.1, 3 The magnitude of the effects ranges from a complete loss of function or acquisition of a new function to very subtle modulations, which are observed in the majority of cases reported.3 Gene regulation through alternative splicing is indeed more versatile than through promotor activity, which modulates the amount of available transcripts without generating novel mRNAs. Microarray experiments indicate that 74% of all human genes are alternatively spliced,7 suggesting that this process is not merely the exception, but rather the rule. The functional changes resulting from alternative pre-mRNA splicing are unknown in most cases, and it will be a major task for the future to assign a specific function to each protein being generated by alternative pre-mRNA splicing. However, this challenge is hindered by the fact that the vast majority of changes caused by alternative splicing are subtle and difficult to detect.3 One exception is ion channels whose electrophysiological properties can be measured. One notable example is the large-conductance Ca2+- and voltage-activated K+ channel (BK). The BK mRNA has over 90 splice isoforms. In addition to different Ca2+ and phosphorylation sensitivities of the splice isoforms, they are also sensitive to electrical potential and are regulated by protein kinases, all depending on the splice isoform.8, 9 Another example is the T-type Ca2+ channels (CaV3), some of which compare more than 30 splice isoforms. The T-type Ca2+ channels are implicated in diverse processes including action potential firing, hormone secretion, smooth muscle contraction, myoblast fusion and fertilization.10 This functional heterogeneity may be explained by alternative splicing of ion channel subunits affecting kinetics and voltage dependency.11 Messenger RNAs can be modified by alternative splicing in several ways. The major splice events are exon skipping, 3′ or 5′ alternative splice sites, retained introns and mutually exclusive exons (Figure 1B). Exon retention or skipping is the prevalent form of alternative splicing. Here, a specific exon is included in the mRNA or spliced out. This can give rise to a protein with completely new function, to loss of function or to subtle modification of the protein. In the case of the RAC1, a 19 amino acids insertion leads to decreased GTPase activity and reduced affinity to GDP.12 RAC1 is a regulator of the endothelial barrier,13, 14 and alternatively spliced RAC1 isoforms play an important role in breast cancer cell migration. If one exon is skipped and replaced by a new exon, it is called mutually exclusive exon. The myokine IL-15 is modified by exchanging exon 5 with an alternative exon 5′. This alternative exon 5′ incorporates an early stop codon into the mRNA leading to a shorter variant of IL-15 that lacks the hydrophobic domain. This IL-15 is not secreted and retained in the cytosol.15 The non-secretable IL-15 suppresses IL-15 gene transcription, implying a novel autocrine regulatory mechanism for myokine gene expression by alternative splicing. There are multiple other alternative splicing events affecting the myokine signalling pathway. Myokines such as IL-15, myostatin and myonectin are required for myogenesis16 and restructuring of skeletal muscle in response to exercise training.17 PGC-1α is a good example for the modification of protein function using an alternative 3′ splice site. Alternative splicing results in a longer exon leading to the PGC-1α4 splice isoform, which is expressed in skeletal muscle, promoting physiological muscle hypertrophy by inducing IGF-1 expression and reducing the levels of myostatin. There are indications that PGC-1α may be involved in muscle regeneration from injury.18, 19 Changes caused by alternative splicing in one particular tissue can also bear on other tissues and have systemic effects by secreting circulating factors (myokines). Thus, skeletal muscle overexpression of PGC-1α4 results in browning of adipose tissue.16, 20 Alternative splicing does not only effect exons, but can also lead to modified mRNAs containing intronic sequences. The predominant form here is the retained intron. In this case, an intron is not spliced out from the mRNA. A retained intron usually leads to degradation of the mRNA by a process called nonsense-mediated decay. Myostatin is effected by a different way of intron modification called the cryptic intron. The myostatin mRNA contains an intron that is usually not excluded from the mRNA. This predominant myostatin plays a complex regulatory role in skeletal muscle homeostasis21 and is known as a negative regulator of muscle growth. However, the secondary myostatin splice isoform with the excluded cryptic intron has an inverse function, stimulating myogenesis through the induction of myogenic regulatory factors.22 Realizing the enormous diverse functions arising from alternative splicing, it is not surprising that abnormal regulation of alternative splicing represents a common paradigm in human disease. Disease-promoting events are frequently caused by mutations in regulatory mRNA sequences and regulatory factors.3 Mutations in the oestrogen receptor are associated with breast cancer. A point mutation in the human oestrogen receptor gene, giving rise to an abnormal oestrogen receptor mRNA, was discovered in human breast cancer biopsy samples.23 The point mutation generates a new splice donor site leading to the insertion of 69 nucleotides that are erroneously recognized as exonic sequence by the spliceosome. Oestrogen receptors have more than 10 splice isoforms, and it is intriguing to ask how these splice isoforms affect other functions of the oestrogen receptors, for example in the cardiovascular system.24 Another example of a disease-associated splice event is the dopamine D3 receptor. Nagai et al25 detected a decrease in the dopamine D3 receptor mRNA expression in the context of Parkinson's disease.26 Alternative splicing introduced a frameshift leading to a loss of the dopamine binding region. Several protein isoforms resulting from alternative splicing have come into focus for experimental therapeutic intervention. Small molecule modulators of alternative pre-mRNA splicing have recently emerged as promising novel tools for cancer therapy in preclinical studies.27 Targeting the spliceosome component SF3B1 has been used to suppress tumour growth. Inhibition of SF3B1 generates special isoforms of Cyclin A2 and Aurora A kinase, slowing down cell cycle progression, and, in this way, impedes cancer growth. To increase target site specificity, it may become necessary to differentiate between splice isoforms such as in the case of Claudin18. The human Claudin18 gene makes use of two alternative first exons, resulting in isoforms CLDN18.1 and CLDN18.2. The claudins are the most important part of the tight junctions28 and essential for cell-cell contact. CLDN18.2 is preferentially expressed in gastric tissue where it contributes to the mucosal barrier. Claudin18.2 is upregulated in gastric cancer, which has prompted to the development of a recombinant monoclonal antibody against CLDN18.2 for potential diagnostic and therapeutic use.29 Alternative pre-mRNA splicing has the potential to generate a multitude of proteins with not only one but possibly two to hundreds of different functions from a single gene. A detailed knowledge of the different protein functions is important for a more complete understanding of human physiology and for coping with the obstacles of personalized medicine. There is no conflict of interest.