What Are Poison Exons?

Poison exons are specific exonic sequences within a gene that are alternatively spliced into pre-mRNA transcripts but introduce premature termination codons (PTCs) or disrupt normal protein synthesis. When included in the mature mRNA, these exons typically cause the transcript to become a target for nonsense-mediated mRNA decay (NMD) or lead to the production of a non-functional or truncated protein.

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Key Characteristics of Poison Exons

1. Premature Termination Codons (PTCs):
   • Poison exons often contain stop codons in positions that would prematurely terminate translation, making the resulting mRNA defective.
2. Negative Regulatory Role:
   • They are thought to play a role in regulating gene expression levels by controlling the stability of the mRNA. By inducing NMD, the inclusion of poison exons can lower the abundance of functional mRNA.
3. Alternative Splicing:
   • Poison exons are included or excluded from the final transcript depending on the splicing machinery and regulatory signals in the cell.
4. Conservation:
   • Many poison exons are evolutionarily conserved, suggesting that they play an important regulatory or adaptive role.
5. Functional Targets:
   • Poison exons are often found in genes encoding splicing factors, transcription regulators, or other proteins involved in RNA processing, suggesting a role in fine-tuning RNA-related processes.

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Biological Functions of Poison Exons:

1. Gene Expression Regulation:
   • Poison exons help regulate the expression of specific genes by influencing the degradation of mRNA via NMD. This is a mechanism for maintaining proper protein levels.
2. Splicing Factor Control:
   • Many splicing factors (e.g., SR proteins and hnRNPs) have their own transcripts regulated by poison exons. This creates a feedback loop where splicing factors regulate their own production to maintain cellular homeostasis.
3. Cellular Stress Response:
   • Poison exon inclusion can be modulated in response to cellular stress or environmental changes, altering gene expression patterns accordingly.
4. Developmental Regulation:
   • During development, poison exons can control the timing and level of expression of key regulatory genes.

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Clinical Relevance:

1. Genetic Disorders:
   • Aberrant splicing of poison exons can lead to diseases caused by dysregulated gene expression, such as some forms of cancer and neurodegenerative diseases.
2. Cancer:
   • Dysregulation of splicing factors controlled by poison exons has been implicated in the proliferation of certain cancers. For example, altered splicing of poison exons in tumour suppressor genes or oncogenes can affect tumour progression.
3. Potential Therapeutic Targets:
   • Manipulating the splicing of poison exons (e.g., using antisense oligonucleotides) could restore normal gene expression and serve as a treatment strategy for diseases involving splicing dysregulation.

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Examples of Genes with Poison Exons

1. SRSF2 (Serine/Arginine-Rich Splicing Factor 2):
   • The inclusion of a poison exon in SRSF2 transcripts leads to NMD and regulates the levels of this splicing factor.
2. HNRNPs (Heterogeneous Nuclear Ribonucleoproteins) – Genes encoding hnRNP proteins, such as HNRNPA1, also harbour poison exons. The regulation of these genes ensures balanced RNA processing and alternative splicing. 

3. Transcription Factors

   • Poison exons regulate transcription factors like E2F1 and FOXP1, which are critical for cell cycle progression, apoptosis, and development. Misregulation of these factors can have profound effects, including cancer development.

   3. Genes Involved in Splicing Regulation

   • Many genes that regulate splicing themselves (e.g., RBM5, RBM10) include poison exons, creating a feedback loop to tightly control their own expression levels. 
   • RBM5 (RNA-Binding Motif Protein 5):
     • Poison exon splicing regulates the production of isoforms involved in apoptosis and cancer.

   4. Neurological Genes

   • Poison exons are found in genes involved in neuronal development and function, such as PTBP1 (Polypyrimidine Tract Binding Protein 1). This is particularly important during neuron differentiation, where alternative splicing patterns change dynamically.

   5. Immune System Genes

   • Poison exons are also implicated in immune system regulation, including in T-cell receptor genes and genes encoding components of the immune signalling pathways.

   6. Oncogenes and Tumour Suppressors

   • Certain oncogenes and tumour suppressor genes, such as MDM2 and TP53, can contain poison exons that regulate their expression levels to maintain cellular homeostasis and prevent tumorigenesis.

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Research Implications

• Poison exons represent an important regulatory mechanism for gene expression and RNA metabolism. Ongoing research into their roles, especially in diseases, is uncovering new layers of complexity in gene regulation. By understanding how poison exons are spliced, scientists can develop novel therapeutic strategies for diseases linked to splicing defects.

Exploiting poison exons for clinical benefits involves targeting their splicing, inclusion, or exclusion to modulate gene expression in ways that can treat or prevent diseases. Since poison exons regulate mRNA stability and protein levels, they offer an opportunity to control gene expression precisely, especially in conditions where splicing is dysregulated or where protein overproduction or deficiency is implicated. Here are key strategies for clinical exploitation:

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1. Therapeutic Strategies Targeting Poison Exons

A. Antisense Oligonucleotides (ASOs)

• Mechanism: ASOs are short, synthetic nucleotides that bind to specific RNA sequences. They can block or promote the inclusion of poison exons by modulating the binding of splicing factors.
• Applications:
  • Enhancing Poison Exon Exclusion:
    • In diseases where a toxic protein is overexpressed, such as in certain cancers, ASOs can suppress the inclusion of poison exons, restoring NMD and reducing harmful protein levels.
    • Example: Downregulating oncogenes or splicing regulators.
  • Promoting Poison Exon Inclusion:
    • In diseases caused by overproduction of specific proteins, ASOs can increase poison exon inclusion to degrade the mRNA through NMD.
    • Example: Reducing the expression of splicing factors like SRSF1, whose overexpression is linked to cancer.
• Advantages: Highly specific, modifiable, and widely studied (FDA-approved therapies like nusinersen for spinal muscular atrophy use ASOs).

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B. CRISPR-Based Splicing Modulation

• Mechanism: CRISPR-Cas9 or CRISPR-Cas13 can be used to edit sequences around poison exons or directly target the splicing machinery to alter exon inclusion or exclusion.
• Applications:
  • Gene Editing for Poison Exon Inclusion:
    • Introducing mutations in splicing enhancers near poison exons to trigger NMD for genes that cause disease.
  • Permanent Correction of Aberrant Splicing:
    • Correcting mutations that disrupt normal poison exon regulation in diseases caused by splicing defects.
• Advantages: Permanent solutions with the potential for single-dose therapies.

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C. Small Molecule Modulators

• Mechanism: Small molecules can modulate the activity of splicing factors, influencing poison exon inclusion or exclusion.
• Applications:
  • Regulating Splicing Factors:
    • For diseases like cancer or neurodegenerative disorders, where splicing dysregulation is central, small molecules can restore balanced splicing.
  • Targeting Specific Splicing Events:
    • Compounds like pladienolide or spliceostatin A, which inhibit the spliceosome, can be tailored to favor poison exon inclusion or exclusion.
• Advantages: Systemic delivery is feasible, and oral administration could be developed.

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D. RNA Interference (RNAi)

• Mechanism: Small interfering RNAs (siRNAs) can degrade pre-mRNA transcripts containing poison exons.
• Applications:
  • Knockdown of Aberrant Transcripts:
    • Selectively degrading harmful transcripts by enhancing poison exon inclusion and promoting NMD.

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2. Clinical Conditions That Could Benefit from Poison Exon Targeting

A. Cancer

• Overexpression of splicing factors or oncogenes can drive tumor progression.
• Example:
  • Targeting SRSF1 or SRSF2 poison exon splicing can reduce the levels of these splicing factors and slow cancer cell growth.
• Approach:
  • Use ASOs or CRISPR to restore poison exon splicing regulation in oncogenes.

B. Neurodegenerative Diseases

• Alternative splicing plays a critical role in neurodegenerative disorders like amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
• Example:
  • Poison exon regulation in genes like TAU or TDP-43 could reduce toxic protein accumulation.
• Approach:
  • Promote poison exon inclusion in transcripts to trigger NMD and decrease protein levels.

C. Splicing Factor Dysregulation Disorders

• Dysregulated splicing factors cause diseases like myelodysplastic syndromes (MDS).
• Example:
  • SRSF2 mutations lead to abnormal splicing and disease progression.
• Approach:
  • Modulate poison exon inclusion in SRSF2 mRNA to regulate its expression.

D. Genetic Disorders

• Many genetic disorders involve splicing defects, where improper poison exon regulation leads to disease.
• Example:
  • Correcting poison exon splicing defects in diseases like Duchenne muscular dystrophy or spinal muscular atrophy.
• Approach:
  • Use ASOs or small molecules to correct splicing and restore functional protein expression.

E. Cardiovascular Diseases

• Alternative splicing of genes involved in heart function can lead to cardiomyopathies.
• Approach:
  • Targeting poison exons in genes regulating heart muscle proteins.

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3. Challenges and Solutions in Exploiting Poison Exons

A. Delivery Issues

• Challenge: Delivering ASOs, siRNAs, or CRISPR therapies to specific tissues can be difficult.
• Solution:
  • Develop tissue-specific delivery systems like lipid nanoparticles or viral vectors.

B. Off-Target Effects

• Challenge: Modulating splicing may unintentionally affect other transcripts.
• Solution:
  • Use advanced bioinformatics tools to design highly specific ASOs or CRISPR guides.

C. Immune Response

• Challenge: Immune activation against RNA or DNA therapies.
• Solution:
  • Modify RNA molecules chemically (e.g., 2′-O-methyl modifications) to reduce immunogenicity.

D. Complex Splicing Networks

• Challenge: Poison exons are part of intricate splicing networks, and altering one may disrupt others.
• Solution:
  • Perform comprehensive studies of splicing networks using RNA sequencing to predict downstream effects.

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4. Emerging Technologies to Enhance Poison Exon Targeting

A. Artificial Intelligence and Machine Learning

• AI tools can predict the impact of poison exon splicing modulation on the transcriptome, allowing for better therapeutic designs.

B. Synthetic Biology

• Engineering synthetic splicing factors or pathways to target poison exons specifically and efficiently.

C. Single-Cell RNA Sequencing

• Enables precise understanding of poison exon regulation in individual cells, especially in heterogeneous diseases like cancer.

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Conclusion

By targeting poison exons, researchers can harness the natural mechanisms of mRNA decay and splicing regulation to treat diseases at the genetic level. Emerging biotechnologies like ASOs, CRISPR, and small molecule splicing modulators are already proving their potential to manipulate poison exons for clinical benefits, offering hope for addressing a wide range of genetic, neurodegenerative, and cancer-related disorders. While challenges remain, the field is rapidly advancing, paving the way for novel, targeted therapies.