Pre-mRNA splicing is a vital process in eukaryotic gene expression and regulation, whereby introns are removed and exons are joined to form mature mRNA molecules. This process is mediated by a complex machinery called the spliceosome, which recognizes and cleaves the splice sites at the boundaries of introns and exons. Pre-mRNA splicing can generate multiple mRNA isoforms from a single gene through alternative splicing, which is a major source of gene complexity and diversity in eukaryotes. However, pre-mRNA splicing is not always sequential, meaning that introns are not necessarily removed in the order of their appearance in the transcript. Nonsequential intron removal, also known as noncanonical or nonordered splicing, is a phenomenon that has been observed in various organisms, from yeast to humans. Nonsequential splicing can affect the splicing outcome and regulation of alternative splicing, as well as the interactions and dynamics of the spliceosome components. Antisense oligonucleotides (AOs) are synthetic nucleic acid molecules that can bind to specific RNA targets and modulate their splicing, stability, or translation. AOs have been developed as a powerful tool to manipulate splicing and treat various diseases caused by splicing defects or mutations, such as Duchenne muscular dystrophy, spinal muscular atrophy, and Huntington's disease. However, the design and efficacy of AOs can be influenced by nonsequential splicing, as the order of intron removal can affect the accessibility and availability of the AO target sites.
The order of intron removal is not random, but rather influenced by various factors that can affect the splicing efficiency and outcome. These factors can be classified into two categories: cis-acting and trans-acting factors. Cis-acting factors are the intrinsic features of the pre-mRNA sequence or structure, such as intron length, position, sequence, structure, and splicing signals. Intron length is one of the most important cis-acting factors that affects the order of intron removal. Shorter introns are removed faster and earlier than longer introns, as they require less time and energy for spliceosome assembly and catalysis. Intron position is another cis-acting factor that affects the order of intron removal. Introns closer to the 5' end of the transcript are removed earlier than introns closer to the 3' end, as they are more accessible and exposed to the splicing machinery. Intron sequence and structure are also cis-acting factors that affect the order of intron removal. Intron sequence and structure can influence the recognition and binding of the splicing signals, such as the 5' splice site, the branch point, the polypyrimidine tract, and the 3' splice site, by the spliceosome components. Intron sequence and structure can also affect the stability and folding of the pre-mRNA, which can influence the accessibility and availability of the splicing signals. In general, introns with stronger and more conserved splicing signals and introns with less stable and more linear structures are removed faster and earlier than introns with weaker and less conserved splicing signals and introns with more stable and more complex structures.
Trans-acting factors are the extrinsic factors that interact with the pre-mRNA, such as splicing factors, chromatin modifiers, transcription factors, and RNA-binding proteins. They are the extrinsic factors that interact with the pre-mRNA and affect the order of intron removal. Trans-acting factors include splicing factors, chromatin modifiers, transcription factors, and RNA-binding proteins, which can regulate the splicing process by enhancing or inhibiting the recognition and binding of the splicing signals, by altering the chromatin structure and accessibility of the pre-mRNA, by modulating the transcription rate and elongation of the pre-mRNA, and by forming secondary or tertiary interactions with the pre-mRNA. Generally, trans-acting factors that promote splicing, such as splicing enhancers, histone acetyltransferases, transcription activators, and RNA-binding proteins that stabilize the pre-mRNA, can accelerate and advance the removal of introns. Conversely, trans-acting factors that repress splicing, such as splicing silencers, histone deacetylases, transcription repressors, and RNA-binding proteins that destabilize the pre-mRNA, can delay and postpone the removal of introns. However, trans-acting factors are not the only determinants of splicing order, as some trans-acting factors can have different effects on different introns within the same transcript, depending on the context and the interaction with other factors. For example, the splicing factor SRSF, which is generally a splicing enhancer, can promote the removal of the first intron but inhibit the removal of the second intron of the human FOS gene. This suggests that other factors, such as intron length, position, sequence, structure, and other trans-acting factors, can modulate the effect of trans-acting factors on splicing order.
| Factor | Description | Example |
|---|---|---|
| Intron length | The size of the intron in nucleotides | Shorter introns are usually removed faster and earlier than longer introns. |
| Intron position | The location of the intron in the transcript | Introns closer to the 5' end are usually removed earlier than introns closer to the 3' end. |
| Intron sequence | The nucleotide composition and conservation of the intron | Introns with stronger and more conserved splicing signals are usually removed faster and earlier than introns with weaker and less conserved splicing signals. |
| Intron structure | The secondary and tertiary folding of the intron | Introns with less stable and more linear structures are usually removed faster and earlier than introns with more stable and more complex structures. |
| Trans-acting factors | The extrinsic factors that interact with the intron | Trans-acting factors that promote splicing can accelerate and advance the removal of introns, while trans-acting factors that repress splicing can delay and postpone the removal of introns. |
Table 1. Comparison of factors affecting the order of intron removal
To study the order of intron removal, various methods have been developed and applied based on different types of RNA-seq technologies. These methods can be broadly classified into two categories: RT-PCR-based and RNA-seq-based methods. RT-PCR-based methods use reverse transcription and polymerase chain reaction to amplify and detect specific splicing intermediates or products, such as lariat-exon or exon-exon junctions, that reflect the order of intron removal. RNA-seq-based methods use high-throughput sequencing to capture and quantify the abundance of splicing intermediates or products, either by using short reads or long reads. Each method has its own advantages and disadvantages, such as sensitivity, specificity, throughput, and resolution.
RT-PCR-based methods are the traditional and widely used methods for studying the order of intron removal. These methods rely on the design of specific primers that target the splicing intermediates or products of interest, such as lariat-exon or exon-exon junctions, and the use of reverse transcription and polymerase chain reactions to amplify and detect them. RT-PCR-based methods have several advantages, such as high sensitivity, specificity, and accuracy, as well as low cost and easy implementation. However, RT-PCR-based methods also have several limitations, such as low throughput, scalability, and coverage, as well as the requirement of prior knowledge of the splicing events and the primer design.
RNA-seq-based methods are emerging and promising methods for studying the order of intron removal. These methods rely on the use of high-throughput sequencing to capture and quantify the abundance of splicing intermediates or products, either by using short reads or long reads. Short reads are typically 50-300 bp in length and long reads are typically >1 kb in length. RNA-seq-based methods have several advantages, such as high throughput, scalability, and coverage, as well as the ability to discover novel splicing events and patterns. However, RNA-seq-based methods also have several challenges, such as high cost, complexity, and error rate, as well as the need for sophisticated bioinformatics tools and pipelines.
| Method | Advantages | Disadvantages | Examples |
|---|---|---|---|
| RT-PCR-based methods | High sensitivity, specificity, and accuracy; low cost and easy implementation | Low throughput, scalability, and coverage; require prior knowledge and primer design | RT-PCR-based methods have been used to study the order of intron removal of the human FOS, CD44, and SMN2 genes. |
| RNA-seq-based methods | High throughput, scalability, and coverage; can discover novel splicing events and patterns | High cost, complexity, and error rate; require sophisticated bioinformatics tools and pipelines | RNA-seq-based methods have been used to study the order of intron removal of human, mouse, zebrafish, Arabidopsis, and Euglena gracilis transcripts. |
Table 2. Comparison of RT-PCR-based methods and RNA-seq-based methods
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Nonsequential splicing can have important implications for splice-manipulating therapies, which use antisense oligonucleotides (AOs) to modulate the splicing of target genes and treat various diseases. AOs are synthetic nucleic acid molecules that can bind to specific RNA sequences and alter their splicing, stability, or translation. AOs have been successfully applied to treat several diseases caused by splicing defects or mutations, such as Duchenne muscular dystrophy, spinal muscular atrophy, and Huntington's disease.
However, the design and efficacy of AOs can be influenced by nonsequential splicing, as the order of intron removal can affect the accessibility and availability of the AO target sites. For example, if an intron is removed before an exon that is targeted by an AO, the AO may not be able to bind to the exon and induce its skipping. Conversely, if an intron is removed after an exon that is targeted by an AO, the AO may bind to the exon and prevent its inclusion. Therefore, understanding the order of intron removal and its impact on splicing regulation is essential for optimizing the design and delivery of AOs.
Nonsequential splicing can also provide opportunities for splice-manipulating therapies, as it can enable the skipping of multiple exons or exon blocks with a single AO. For example, in Duchenne muscular dystrophy, a disease caused by mutations in the dystrophin gene, some introns are removed before the adjacent exons, creating exon blocks that can be skipped together with a single AO. This can increase the efficiency and feasibility of restoring the reading frame and producing a functional dystrophin protein.
Therefore, nonsequential splicing is a phenomenon that needs to be considered and exploited for splice-manipulating therapies. However, there are still many challenges and limitations in studying and manipulating nonsequential splicing, such as the lack of comprehensive and accurate methods, the variability and context-dependence of splicing order, and the potential side effects and toxicity of AOs. Further research and development are needed to overcome these challenges and improve the safety and efficacy of splice-manipulating therapies.
