What Is Alternative Splicing and Why Is It Important?

Genes contain the instructions for building proteins, but these instructions are not always used in a straightforward way. Through alternative splicing, cells generate multiple protein variants from a single gene, increasing complexity without requiring additional genetic material.

This process is essential for development and cellular function, influencing tissue differentiation, immune responses, and more. Understanding alternative splicing provides insight into biological diversity and how errors in this mechanism contribute to disease.

Stages Of Splicing

Before a gene’s instructions can be translated into a protein, its precursor messenger RNA (pre-mRNA) undergoes splicing. This process removes non-coding sequences (introns) while joining coding regions (exons). It takes place within the spliceosome, a ribonucleoprotein complex composed of small nuclear RNAs (snRNAs) and associated proteins. The spliceosome assembles in steps, recognizing specific nucleotide sequences at exon-intron boundaries to ensure accurate excision and ligation. Errors can lead to faulty transcripts, potentially producing harmful proteins.

Splicing begins with the recognition of conserved splice sites at the 5′ and 3′ ends of introns. The 5′ splice site, typically marked by a GU sequence, is identified by U1 snRNA, while the 3′ splice site, ending in AG, is recognized by U2 auxiliary factors. A branch point sequence upstream of the 3′ splice site is bound by U2 snRNA, positioning the intron for removal. These interactions set the stage for the catalytic steps that follow.

Once splice sites are identified, the spliceosome undergoes rearrangements, bringing reactive groups into proximity for the first transesterification reaction. The branch point adenosine attacks the 5′ splice site, forming a lariat intermediate. In the second reaction, the freed 3′ hydroxyl group of the upstream exon attacks the 3′ splice site, leading to exon ligation and intron release. The excised intron is degraded, and the mature mRNA is exported for translation.

Types Of Alternative Splicing

Alternative splicing allows a single gene to produce multiple mRNA variants by selectively including or excluding specific exons or splice sites. This expands protein diversity without additional genetic material. Several patterns of alternative splicing influence gene expression and protein structure.

Exon Skipping

Exon skipping, the most common form of alternative splicing in mammals, removes a specific exon along with its flanking introns. This can alter protein function, stability, or interactions.

For example, the fibronectin gene (FN1) undergoes exon skipping to produce isoforms that affect cell adhesion and migration. The inclusion or exclusion of exon EDA influences fibronectin’s ability to bind integrins, key players in extracellular matrix interactions. In the dystrophin gene (DMD), exon skipping has been explored as a therapy for Duchenne muscular dystrophy (DMD). By inducing the exclusion of specific exons, researchers aim to restore a partially functional dystrophin protein. Eteplirsen, an exon-skipping antisense oligonucleotide, received FDA approval in 2016 for this purpose.

Mutually Exclusive Exons

In mutually exclusive exon splicing, only one exon from a set is included in the final transcript, while others are omitted. This ensures distinct protein isoforms with different structural or functional properties. Splicing enhancers and silencers regulate which exon is selected.

The tropomyosin gene (TPM) provides a clear example, producing muscle-specific isoforms through mutually exclusive exon selection. This allows for tissue-specific functional adaptations. Likewise, the calcium channel gene CACNA1 undergoes alternative splicing that alters ion conduction properties, affecting neuronal excitability. Disruptions in this process have been linked to neurological disorders such as epilepsy and autism.

Alternative 5′ Or 3′ Splice Sites

Alternative splicing can also involve different splice sites at the 5′ or 3′ ends of an exon, altering exon length. This fine-tunes protein domains, impacting stability, localization, or interactions. Splice site selection is influenced by regulatory elements and RNA-binding proteins.

The Bcl-x gene (BCL2L1) exemplifies this mechanism, producing two isoforms with opposing roles in apoptosis. The longer Bcl-xL isoform promotes cell survival, while the shorter Bcl-xS induces programmed cell death. Their relative expression is tightly regulated, with implications for cancer progression, as an imbalance favoring Bcl-xL can contribute to tumor resistance.

Similarly, alternative 3′ splice site selection in the FAS gene generates different Fas receptor isoforms, which regulate apoptosis. The full-length isoform promotes cell death, while a shorter, soluble variant inhibits it. Dysregulation of this splicing event has been implicated in autoimmune diseases and cancer.

Role In Protein Diversity

Alternative splicing enhances proteome complexity by enabling a single gene to produce multiple protein isoforms. Unlike mutations, which require permanent DNA changes, splicing allows dynamic regulation of protein function without altering genetic code. By including or excluding exons, cells generate proteins with distinct biochemical properties, affecting enzymatic activity, binding affinity, or localization.

A striking example is titin, the largest known human protein, which plays a key role in muscle elasticity. The TTN gene undergoes extensive alternative splicing to produce isoforms that vary in size and mechanical properties. In cardiac muscle, different titin variants adjust contractile stiffness in response to physiological demands. Similarly, in neurons, alternative splicing of scaffolding proteins like PSD-95 influences synaptic architecture, affecting learning and memory.

Beyond structural diversity, alternative splicing also shapes signaling pathways by generating protein isoforms with opposing functions. The FGFR2 gene, for instance, encodes fibroblast growth factor receptors that regulate cell proliferation and differentiation. Two major isoforms, FGFR2-IIIb and FGFR2-IIIc, arise from alternative splicing and exhibit distinct ligand-binding affinities. The IIIb variant is expressed in epithelial cells, while IIIc is found in mesenchymal tissues, directing tissue-specific responses to growth factors.

Impact On Gene Regulation

Alternative splicing modulates gene expression by altering protein availability, stability, and activity. This allows cells to adapt to developmental cues, environmental stimuli, and metabolic demands. Splicing particularly impacts transcription factors and RNA-binding proteins, which themselves undergo alternative splicing to produce isoforms with distinct regulatory roles.

A well-documented example is the splicing of the REST transcription factor, which regulates neuronal gene expression. Depending on the splice variant, REST can either repress or permit neuronal gene transcription, influencing neurodevelopment. Similarly, alternative splicing of FOXP1 generates isoforms with different DNA-binding affinities, affecting gene networks involved in cardiac development and neurogenesis.

Association With Disease States

Errors in alternative splicing contribute to many diseases by producing dysfunctional proteins or disrupting regulatory isoforms. Genetic disorders, neurodegenerative diseases, and cancers often involve splicing defects caused by mutations affecting splice site recognition or regulatory elements. These errors can lead to exon skipping, aberrant splice site selection, or the inclusion of cryptic exons.

Spinal muscular atrophy (SMA) provides a notable example. This neurodegenerative disorder is caused by mutations in the SMN1 gene, but its severity is influenced by splicing of the nearly identical SMN2 gene. The preferential skipping of exon 7 in SMN2 results in a truncated, less functional protein. Antisense oligonucleotides, such as nusinersen (FDA-approved in 2016), target this defect to promote exon inclusion and restore functional SMN protein levels.

In cancer, splicing alterations in tumor suppressor genes like TP53 or oncogenes such as BCL2 influence tumor progression by disrupting apoptosis and cell cycle regulation. Certain malignancies, including glioblastomas and leukemias, exhibit distinct splicing patterns that affect tumor aggressiveness and therapy response. Understanding these alterations has led to the development of splicing-modulating drugs, such as small molecules targeting the spliceosome, offering new paths for precision medicine.