Alternative splicing (AS) or differential exon usage (DEU) is a regular process af- ter gene expression and it contributes to the diversity of the genome by generating multiple protein isoforms. According to recent studies, the majority (92-94%) of all human multi-exon genes undergo AS and the brain, especially the neocortex, has the highest number of AS events compared to other tissues.
Transcription of mRNAs from DNA produces nascent mRNA (pre-mRNA) molecules that should be further processed in order to be functional. These post- transcriptional modifications include 5’ capping, 3’ polyadenylation and RNA splicing. While 5’ capping and 3’ polyadenylation serve to protect ends of pre- mRNAs from attacks of ribonucleases, RNA splicing is required in order to obtain final mRNA code that will be translated into a protein. This step is required be- cause unlike other prokaryotic mRNAs, eukaryotic mRNAs are discontinuous with exon regions that will be translated and with noncoding intron regions that need to be removed.
RNA splicing is carried out by large RNA-protein complexes (RNPs) called spliceosomes. The conventional U2-dependent spliceosome is composed of five small nuclear RNPs (snRNPs) U1, U2, U4, U5 and U6 and many accessory pro- teins (Staley and Guthrie, 1998; Jurica and Moore, 2003). Each snRNP molecule contains an RNA component (snRNA), a set of seven Sm proteins (B/B’, D3, D2, D1, E, F, and G) and varying number of other accessory proteins.
Splicing is carried out by a series of snRNP and mRNA interactions. Certain snRNPs can bind to conserved sequences on mRNAs through base pairing with their RNA components. These sequences, demonstrated in Fig. 1.1, are; 5’ and 3’ splice sites, branch point sequence (BPS) and polypyrimidine tract (PPT). 5’ and 3’ splice sites are found at the beginning and at the end of introns, defining the exon-intron boundaries. They can change according to the type of intron but most commonly occurring U2-type introns have GU at their 5’ site and AG at their 3’ end. Branch point sequence can be found anywhere from 18 to 40 nucleotides upstream of the 3’ end of an intron. It is not very well conserved and has a typical sequence ”YNYYRAY”, where Y indicates a pyrimidine, N is any nucleotide, R indicates any purine, and A stands for adenine. Polypyrimidine tract lies between BPS and 3’ splice site of an intron, located 5 to 40 nucleotides upstream of the 3’ end of an intron. It is rich in terms of pyrimidine nucleotides, especially uracil, and usually 15–20 base pairs long.
If an intron is short (<200–250 nts), spliceosome machinery forms across this intron. At the early stages of splicing, U1 snRNP binds to 5’ splice site (GU) and other accessory proteins SF1/mBBP and U2AF bind to the BPS and PPT, respectively. At this step spliceosome is called complex E. Then, U2 snRNP binds to adenine base of the branch point sequence (BPS), forming complex A (prespliceosome). A trimer containing U4/U5, U6 interacts with both U1 and U2, forming complex B (precatalytic spliceosome). Then the release of U1 and U4 allows other components of the spliceosome, especially U6 snRNP, to come into close position to 5’ splice site. The complex which is now called complex B2 is activated and can carry out splicing reaction. Then the first catalytic step occurs: 2’OH group of Adenine at the BPS which is brought to closer proximity to the 5’ splice site attacks phosphodiester bond of Guanine at the 5’ splice site. As a result, intron is cleaved from the 5’ splice position, releasing the first exon that it is bound to while at the 3’ splice site it is still bound to the second exon as a loop structure called lariat. Then the second catalytic step occurs: Free 3’ OH group of the released first exon attacks the phosphodiester bond at the 3’ splice site (AG), releasing the lariat which is rapidly degraded. As a result of this process, two exons are ligated to each other. At the end, spliceosome is disassembled until next splicing reaction.
However, if an intron is longer (> 200-250 nts) like the most of eukaryotic introns, the spliceosome machinery is first formed on an exon through a process called exon definition. In this case, U1 binds to the 5’ splice site while U2AF interacts with the PTT sequence of the upstream intron, defining the beginning and the end of exon. Then, U2 is recruited to the BPS of upstream intron. Fi- nally, with the recruitment of other accessory proteins, exon-defined spliceosome is stabilized on exon but in order to cleave an intron, 5’ splice site of spliceo- some machinery should interact with the downstream 3’ splice site of the same intron. This transition from exon-defined to intron-defined spliceosome complex is currently not well understood
However, if an intron is longer (> 200-250 nts) like the most of eukaryotic introns, the spliceosome machinery is first formed on an exon through a process called exon definition. In this case, U1 binds to the 5’ splice site while U2AF interacts with the PTT sequence of the upstream intron, defining the beginning and the end of exon. Then, U2 is recruited to the BPS of upstream intron. Fi- nally, with the recruitment of other accessory proteins, exon-defined spliceosome is stabilized on exon but in order to cleave an intron, 5’ splice site of spliceo- some machinery should interact with the downstream 3’ splice site of the same intron. This transition from exon-defined to intron-defined spliceosome complex is currently not well understood.
There is an alternative spliceosome which uses different snRNPs than the con- ventional one and cleaves the minor class of introns. Introns with splice sites 5’GU- 3’AG comprise the majority of introns but there are introns with splice sites 5’AU-3’AC and 5’GU-3’AG. These minor classes of introns are spliced by an alternative spliceosome containing U11 and U12 snRNPs, and therefore called U12-dependent spliceosomes. Splicing takes place during the transcription process in order to ensure ordered removal of introns as they are released from the transcription complex but it does not always produce a same mature mRNA from the same transcript. There are two general types of RNA splicing. One is called constitutive splicing through which all introns are removed and all exons are ligated together to form a mature mRNA. The other is called alternative splic- ing, and as it name implies in this type of splicing, some exons and introns can be included and/or excluded in different combinations, creating diverse splice vari- ants from one transcript. This alternative splicing process is thought to evolve in order to increase protein diversity in complex organisms. Although gene numbers differ little across different species, the protein diversity varies much more due to posttrancriptional modifications including splicing, probably due to increased intron length and number as species become complex.
Types of Alternative Splicing
There are several types of alternative splicing; cassette exon, intron retention, mutually exclusive exon, alternate 3’ and 5’ splice sites, mutually exclusive 3’ and 5’ untranslated regions (UTRs). Cassette exon events occur when one or more exons are skipped while mutually exclusive alternative splicing events result when pre-mRNA cannot contain both at the same time but can contain each separately. If different 5’ and 3’ competing splice sites are available, one of them can be alternatively selected over others. Mutually exclusive 5’ UTRs occurs when alternative promoters alter the transcription start site, therefore the first exon of pre-mRNA. Similarly, alternative polyadenylation sites can alter the transcription end site (the last exon), and therefore 3’ UTR. Intron retention, as it nam implies, occurs when one or more introns are not removed but kept in the pre- mRNA.
Regulation of Alternative Splicing
Alternative splicing is regulated by several factors including the strength of splice sites, cis-regulatory sequences on pre-mRNAs and trans-acting factors. If an in- tron contains conserved splice site sequences it can be easily detected by spliceo- some machinery and it is cleaved almost every time, resulting in constitutive splicing. However, introns containing weak, i.e. non-conserved, splice sites need other factors like cis-regulatory sequences and trans-acting factors in order to be stably recognized by spliceosome.
Cis-regulatory sequences can be found on either exons or introns, and can act as either silencers or activators. Exonic splicing enhancers (ESEs) or exonic splicing silencers (ESSs) serve to facilitate or inhibit the retention of exons in which they reside, respectively. Similarly, intronic splicing enhancers (ISEs) and intronic splicing silencers (ISSs) serve to facilitate or inhibit the retention of exons from intronic regions. They carry out these functions by recruiting trans-acting factors (RNA binding proteins (RBPs)) which can either activate or suppress the activity of spliceosome.
ESEs are found on nearly all exons and can contain varying range of sequences. Generally, ESEs function by recruiting SR family of proteins (trans-acting factors). These proteins bind to ESEs with their one terminal while facilitating the binding of accessory proteins that can promote spliceosome assembly with their other terminal called RS domain. On the other hand, ESSs bind to hnRNP family of proteins and they may contain several different sequences that can bind to RNA. They can inhibit splicing in various ways including preventing U1 and U2 interaction and displacing snRNP on exons.
There are several sequences and functions for ISEs: clusters of guanine (either 3 or more) can facilitate the recognition of nearby splice sites; CA repeats can facilitate splicing of upstream exons; UGCAUG hexanucleotides or sometimes variations of it are found in the introns downstream of neuron-specific exons. Similar to ESEs ISSs function by recruiting hnRNP family of proteins. Both intronic elements (ISEs and ISSs) contain sequences that can bind to tissue- specific splicing factors.
Intron retention is also regulated by the splicing regulatory elements (SREs) mentioned above. For example, 5’ splice site like sequences on an exon can promote the retention of downstream intron. Also G clusters on ISEs can regulate intron retention by facilitating splicing of retained introns on some gene.
SREs regulate alternative splicing events in a context dependent manner. Their function can change according to their location on mRNAs. For exam- ple, G clusters when they are on introns facilitate splicing; however, when they are located on exons they inhibit splicing. Also SR family of proteins can change their function according to their distance to nearest splice site. Consid- ering the complexity and size of spliceosome, this is reasonable since the activity of these trans-acting factors will be affected according to their distance to the spliceosome complex. For example, G cluster binding hnRNP family of proteins facilitates splicing when they bind to G clusters located at the downstream of 5’ splice sites, but prevents splicing when they bind to G clusters on exons. Also, considering the abundance of the SRE elements across the genome, not all SRE elements can be recognized by trans-acting factors and deciding which SRE elements are recognized and what factors contribute to it is still unknown. How- ever, one major suspect that can affect SRE function is the secondary structures of mRNAs that can make both SREs and splice sites more readily accessible. For example, the loop structure of exon 10 of tau gene affects its splicing by reveal- ing or hiding the 5’ splice site adjacent to this exon. Finally, the ultimate activity of SREs are affected by the availability of trans-acting factors which are most probably the result of tissue and cell type specific splicing patterns.
The regulation of splicing can be also affected by transcription machinery since many splicing events are thought to occur cotranscriptionally. It is found that alternative splicing of some genes is affected by the mutated RNA polymerase II that has a slow elongation rate. Therefore, it seems the splicing regulation mechanisms are complex with many different factors involved in it.
These regulation processes are both temporarily and spatially regulated. For example, among all genes expressed in the brain 0.1% of them exhibit alternative splicing differences only across different regions of the brain, 19.5% of these genes exhibit alternative splicing differences only across different developmental periods and 70.6% exhibits both region and time specific expression. These time and region specific alternative splicing is achieved by the expression of splicing regulatory molecules to be expressed in a timely and tissue specific manner. What affects the expression of these splicing regulatory molecules is probably both environmental and genetic factors, which are still not well known.