The instructions for life are stored in deoxyribonucleic acid (DNA) within the nucleus of every cell. To make a protein, a segment of DNA is first transcribed into a precursor messenger RNA (pre-mRNA) molecule. This pre-mRNA is an unprocessed transcript containing the full set of coded instructions. Before it can be translated into a functional protein, the pre-mRNA must undergo significant processing, including a complex biochemical reaction called splicing. Splicing removes non-coding sequences and joins the remaining coding segments, determining the blueprint for the final protein product.
The Foundation: Understanding Alternative Splicing
Genes are composed of coding segments called exons and non-coding intervening sequences known as introns. During splicing, cellular machinery removes the introns from the pre-mRNA and joins the exons together to form the mature messenger RNA (mRNA) transcript.
Alternative splicing (AS) is a regulatory mechanism allowing a single gene to produce multiple distinct mRNA molecules. Exons from one gene can be joined in various combinations, creating a diversity of related but structurally different proteins (isoforms).
This ability to generate multiple protein isoforms is fundamental to the genetic complexity of higher organisms. Approximately 90 to 95% of human genes undergo alternative splicing, significantly expanding the functional repertoire of the human proteome. This process is tightly regulated in a tissue-specific and developmental stage-specific manner.
The most common form of AS is exon skipping, where an exon is entirely excluded from the final mRNA transcript. Other variations include the use of alternative 5′ or 3′ splice sites, or the retention of an entire intron. For example, the large muscle protein titin produces different forms in fetal versus adult heart tissue, driven by AS events.
Defining Alternative Splicing Modifiers
An alternative splicing modifier is a molecule or compound designed to intentionally influence the natural outcome of the splicing process. These modifiers alter which protein isoforms are produced from a single gene transcript. The goal is often to correct a splicing error causing disease or to engineer a beneficial protein variant.
Modifiers are broadly classified into two categories. The first consists of small molecules, which are typically organic compounds that can be chemically synthesized. These often have the advantage of being orally available and capable of entering cells easily.
The second category is antisense oligonucleotides (AONs), which are short, synthetic fragments of nucleic acid. AONs are designed to be complementary to a specific sequence on the pre-mRNA. Researchers chemically modify these strands to increase their stability and resistance to degradation within the cell.
Mechanisms of Modifier Action
Alternative splicing modifiers employ two primary strategies to alter pre-mRNA processing. The first strategy involves the direct targeting of the pre-mRNA sequence itself, a method commonly used by AONs. An AON is engineered to physically bind to a specific region of the pre-mRNA, often close to a splice site or a regulatory sequence element.
This binding creates a physical block that prevents the large cellular splicing machinery, the spliceosome, from recognizing that particular site. Forcing the machinery to ignore a specific exon is known as exon skipping, which effectively changes the sequence of the resulting mature mRNA. The sequence-specific nature of AONs allows for highly precise modification of a single gene’s splicing pattern.
The second strategy involves targeting the protein components of the splicing machinery, a mechanism frequently utilized by small molecule modifiers. These small molecules interfere with the activity of trans-acting splicing factors. These factors are proteins that bind to the pre-mRNA and help guide the spliceosome to the correct sites. Since these factors are often responsible for tissue-specific splicing, their modulation can have broad effects.
Some small molecules act as “molecular glue,” promoting the proper assembly of the spliceosome on a target pre-mRNA sequence. For instance, certain compounds strengthen the interaction between a weak 5′ splice site and the U1 small nuclear ribonucleoprotein (snRNP), a component of the spliceosome. This mechanism helps fix a naturally occurring structural weakness in the pre-mRNA, ensuring the inclusion of a necessary exon.
Therapeutic Applications
The development of alternative splicing modifiers has emerged as a promising approach for treating numerous genetic disorders where mis-splicing is the underlying cause. Faulty splicing can result in a protein that is truncated, non-functional, or produced at insufficient levels, leading to a disease state. Modifiers counteract these errors by restoring normal protein production or engineering a less harmful, partially functional protein.
A prominent example is Spinal Muscular Atrophy (SMA), a severe neurodegenerative disorder caused by a defect in the SMN1 gene. Patients have a related gene, SMN2, which is mostly mis-spliced, resulting in a non-functional protein. Modifiers like the AON Nusinersen or the small molecule Risdiplam promote the inclusion of exon 7 in the SMN2 transcript. This successfully increases the amount of functional Survival of Motor Neuron (SMN) protein in the patient.
Another application targets Duchenne Muscular Dystrophy (DMD), a muscle-wasting disease caused by mutations that disrupt the reading frame of the dystrophin gene. AONs are used here to induce exon skipping, intentionally bypassing the mutated section of the gene. This modification results in a shorter, partially functional dystrophin protein, effectively converting the severe DMD condition into the milder Becker Muscular Dystrophy phenotype. Research is also exploring these modifiers for various neurological disorders, myotonic dystrophy, and several types of cancer.