Alternative splicing: study genetic complexity through NGS analysis
Introduction
Alternative splicing is a vital mechanism of gene regulation that contributes significantly to the complexity of eukaryotic organisms. By allowing a single gene to produce multiple proteins, alternative splicing expands the diversity of proteins without the need for additional genes. In recent years, Next-Generation Sequencing (NGS) technology has revolutionised the study of alternative splicing, providing detailed insights into its functional implications in health and disease.
This article explores the basics of alternative splicing, its role in genetic diversity, and how NGS analysis has become an invaluable tool for understanding this intricate biological process.
What is alternative splicing?
Alternative splicing is a process during gene expression that allows a single pre-mRNA transcript to be spliced in various ways, producing multiple mature mRNA isoforms from the same gene. This ultimately leads to different protein variants, which may have distinct or even opposing functions within the cell.
In a typical gene, exons (coding regions) and introns (non-coding regions) are transcribed from DNA into pre-mRNA. The introns are removed during RNA splicing, and the exons are joined to form the final mRNA. In alternative splicing, the exons can be rearranged, skipped, or combined in different ways, allowing for greater versatility in protein production.
Types of alternative splicing
Exon skipping: The most common form, where specific exons are excluded from the final mRNA.
Intron retention: An intron remains within the mature mRNA, which can affect protein function.
Alternative 5’ or 3’ splice sites: Different splicing at the 5’ or 3’ ends of exons leads to including shorter or longer exon regions.
Mutually exclusive exons: Only one of two possible exons is included in the final mRNA.
Why is alternative splicing important?
Alternative splicing plays a crucial role in enhancing the functional complexity of an organism's proteome. Despite the relatively small number of genes in the human genome (approximately 20,000), alternative splicing allows for creating over 100,000 distinct proteins.
Functions of alternative splicing:
Protein diversity: Different mRNA isoforms lead to the production of protein variants with different, sometimes tissue-specific, functions.
Gene regulation: Alternative splicing can regulate gene expression by controlling which protein isoforms are produced in response to cellular or environmental signals.
Evolutionary significance: Splicing contributes to species diversity, as organisms with fewer genes compensate by increasing protein variability through splicing mechanisms.
Alternative splicing and disease
Dysregulation of alternative splicing is linked to numerous diseases, including cancer, neurodegenerative disorders (such as ALS and Alzheimer's), and genetic disorders like spinal muscular atrophy (SMA). Mutations in splice sites or splicing regulatory elements can lead to aberrant splicing, resulting in dysfunctional proteins that drive disease development.
In cancer, for instance, aberrant splicing can lead to oncogenes' expression or tumour suppressor genes' inactivation. Understanding how alternative splicing is altered in disease is crucial for developing targeted therapies and diagnostic tools.
NGS analysis: a powerful tool to study alternative splicing
With the advent of Next-Generation Sequencing (NGS), researchers can now analyse alternative splicing at an unprecedented scale. RNA sequencing (RNA-seq) offers high-throughput, deep coverage of transcriptomes, making it possible to identify and quantify splicing events genome-wide.
How NGS helps in studying alternative splicing
Comprehensive transcriptome profiling
NGS provides a comprehensive snapshot of the transcriptome, capturing all the different mRNA isoforms expressed in a cell at a given time. This allows researchers to study alternative splicing events across different tissues, developmental stages, and disease conditions.Quantifying isoform abundance
RNA-seq allows for quantifying mRNA isoforms, revealing which splice variants are expressed at higher or lower levels under certain conditions. This is especially important for identifying isoforms linked to specific diseases or physiological states.Detecting novel splicing events
NGS can uncover novel splicing events that were previously undetectable using traditional methods. Researchers can discover new splice variants with critical functional implications by providing a more complete view of the transcriptome.Understanding splicing regulation
Researchers can use NGS data to study the regulatory elements and trans-acting factors that control splicing decisions. This includes identifying splicing factors (e.g., SR proteins, hnRNPs) and mutations in splicing regulatory regions that lead to aberrant splicing patterns.
Here, you can see some examples of the alternative splicing analysis that NextGenSeek offers.
Applications of NGS in alternative splicing research
1. Cancer research
Aberrant alternative splicing plays a significant role in cancer progression and drug resistance. NGS has been instrumental in identifying cancer-specific splice variants, such as those involved in apoptosis evasion or metastasis. This knowledge can lead to the development of splice-switching therapies targeting these variants.
2. Neurological diseases
Many neurodegenerative diseases are associated with splicing defects. For example, NGS has helped uncover splicing abnormalities in ALS, revealing altered RNA processing in motor neurons. This research opens doors for potential RNA-targeted therapies.
3. Splicing biomarkers for diagnostics
NGS has facilitated the discovery of splice variants that are biomarkers for disease diagnosis and prognosis. Researchers can develop precision medicine approaches that account for an individual's unique splicing landscape by integrating splicing data with clinical information.
4. Therapeutic target discovery
NGS analysis has helped identify potential therapeutic targets by revealing how splicing contributes to disease mechanisms. In diseases like spinal muscular atrophy (SMA), drugs like nusinersen have been developed to correct splicing defects, offering a promising treatment approach.
Challenges in NGS-Based alternative splicing analysis
While NGS has revolutionised splicing research, there are still several challenges to consider:
Data Complexity: Analyzing alternative splicing from RNA-seq data requires sophisticated bioinformatics tools capable of handling large datasets and distinguishing between closely related splice variants.
Noise and Artifacts: Sequencing errors or low expression levels of specific isoforms can introduce noise, making it difficult to identify actual splicing events.
Clinical Translation: While NGS provides valuable insights into splicing mechanisms, translating these findings into clinically relevant therapies requires further validation and robust methodologies.
Conclusion
Alternative splicing is a fundamental process that enhances the complexity of gene expression and proteome diversity. Its role in health and disease has been illuminated through Next-Generation Sequencing (NGS), which provides unprecedented insights into how splicing events are regulated and how they can be altered in various conditions. As research continues, NGS will remain a powerful tool for understanding alternative splicing and harnessing its potential for diagnostics and therapeutics.