Alternative splicing is a regulated process in which a single gene can produce multiple proteins by joining or skipping exons in various combinations.
In humans, alternative splicing increases the diversity of proteins encoded by the genome, contributing to 95% of multi-exonic genes.
What is Alternative splicing?
Alternative splicing is a natural process in which the exons of a single precursor mRNA are joined in different arrangements to produce two or more distinct variants of mature mRNAs.
This mechanism enhances proteome diversity and allows regulation of gene expression after transcription.
Mechanism of Alternative Splicing
The pre-mRNA includes multiple introns along with exons.
During splicing, specific exons are selected to be retained in the mature mRNA.
Splice site selection is regulated by splicing activator and repressor proteins.
Regulatory elements within the pre-mRNA, such as exonic splicing enhancers (ESEs) and exonic splicing silencers (ESSs), also influence splice site selection.
Consensus sequences help define important regions within nuclear introns.
A GU sequence marks the 5′ end of each intron.
Near the 3′ end of the intron lies a branch site, which always includes an adenine (A).
The canonical or consensus sequence around the branch point varies, but it typically includes a polypyrimidine tract followed by an AG dinucleotide at the 3′ end.
The spliceosome, a complex of RNA and proteins, facilitates mRNA splicing and includes small nuclear ribonucleoproteins (snRNPs) U1, U2, U4, U5, and U6. (U3 is not involved in mRNA splicing.)
U1 snRNP binds to the 5′ GU splice site of the intron.
U2 snRNP binds to the branch point adenine, aided by U2 auxiliary factors (U2AF). This forms the spliceosome A complex.
Formation of the A complex is critical in determining which intron and exon ends are selected for splicing.
U4, U5, and U6 snRNPs join to form the tri-snRNP complex.
U6 replaces U1 in binding, while U1 and U4 are released to allow the spliceosome to perform two transesterification reactions.
First, the intron is cleaved at the 5′ splice site and joined to the branch point A through a 2′,5′-phosphodiester bond.
Second, the 3′ splice site is cleaved, and the two exons (upstream and downstream) are joined together by a standard phosphodiester bond.
Types of Alternative Splicing
The mechanism of alternative splicing can occur in several distinct forms:
Constitutive splicing: Introns are spliced out, and exons are joined together in a fixed arrangement.
Mutually exclusive exons: Only one of two possible exons is included in the mature mRNA after splicing.
Exon skipping or cassette exon: Specific exons may either be included in or excluded from the mature mRNA. This is the most common form of alternative splicing (30%) observed in both vertebrates and invertebrates.
Alternative 3′ splice site: Exons are joined using an alternate 3′ splice site, resulting in a variation in the length of the upstream exon.
Alternative 5′ splice site: Exons are joined using an alternate 5′ splice site, affecting the length of the downstream exon.
Intron retention: An intron may either be retained in the mature mRNA or removed. This pattern is most commonly observed in lower metazoans.
Alternative splicing in Eukaryotes
In eukaryotes, a newly transcribed RNA molecule, known as pre-mRNA, is not immediately ready for translation.
It must undergo several processing steps to become mature mRNA suitable for protein synthesis.
These processing steps include:
Addition of a 5′ cap to the beginning of the RNA, known as the 5′ cap.
Addition of a poly-A tail (a sequence of adenine nucleotides) to the 3′ end of the RNA.
Removal of introns through splicing and joining of the remaining exons.
The result of these modifications is a mature mRNA.
This mature mRNA is then transported out of the nucleus to be translated into a protein.
Alternative splicing in Prokaryotes
In prokaryotes, mRNA does not undergo splicing of introns or joining of exons, unlike eukaryotic mRNA.
However, splicing can occur in tRNA (transfer RNA) molecules to produce their functional form.
This involves the attachment of a formalin derivative during the final step of translation.
In this step, the processed tRNA binds to mRNA along with ribosomes to facilitate protein synthesis.
Examples of Alternative Splicing
Alternative splicing is involved in every stage of cancer development.
Dsam gene (Drosophila Down syndrome cell adhesion molecule):
This gene can produce up to 38,016 isoforms from 95 variable exons through alternative splicing.
Mhc gene (Myosin heavy chain):
This gene encodes a protein crucial for muscle cell function.
In Drosophila, the Mhc gene is a classic example of complex alternative splicing.
It contains 30 exons, of which 17 are alternatively spliced.
Except for exon 18 (represented by a single alternative exon), the other alternatively spliced exons are organized into five separate clusters, each containing 2 to 5 exons.
Neurexin gene:
Neurexins are presynaptic cell-adhesion molecules vital for synapse formation and synaptic transmission.
Alternative splicing of neurexin transcripts produces thousands of isoforms, contributing to the functional complexity of neuronal networks.
Importance of Alternative Splicing
The primary function of alternative splicing is to increase the diversity of mRNA expressed from a single genome, enabling the production of a wide variety of proteins.
It helps explain how numerous distinct proteins can be generated from a single gene.
It allows exonic regulatory sequences to maintain a wide range of sequence flexibility without affecting coding functions.
Small changes in the concentration of regulatory proteins can influence protein interactions, leading to different patterns of exon usage.
References
Han, J., Xiong, J., Wang, D., & Fu, X. (2011). Pre-mRNA splicing: timing and localization within the nucleus. Trends in Cell Biology, 21(6), 336–343. https://doi.org/10.1016/j.tcb.2011.03.003
Strachan, T., & Read, A. (2001). Human Molecular Genetics (pp. 372–373). New York: Wiley-Liss.
Nussbaum, R., McInnes, R., Willard, H., Thompson, J., & Thompson, M. (2016). Genetics in Medicine (8th ed., pp. 32–33). Elsevier Inc.
Park, J. W., & Graveley, B. R. (2015). Alternative splicing: mechanisms and regulation. Advances in Experimental Medicine and Biology. Published online April 7, 2015. [PMC Article]
Wang, Y., Liu, J., Huang, B., Xu, Y., Li, J., Huang, L., Lin, J., Zhang, J., Min, Q., Yang, W., & Wang, X. (2015). Mechanism and regulation of alternative splicing. Biomedical Reports, 3(2), 152–158. https://doi.org/10.3892/br.2015.407
