GO:0033120 positive regulation of RNA splicing: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0033120 (positive regulation of RNA splicing) describes any process that increases the frequency, rate or extent of RNA splicing, a co- and post-transcriptional step essential for generating mature mRNAs.
• Positive regulation of splicing is driven by cis-acting RNA elements (exonic/intronic enhancers) and trans-acting factors such as SR proteins and hnRNPs that recruit the spliceosome.
• The stress kinase PKR can be activated by intragenic RNA elements to positively regulate splicing of cellular and viral mRNAs, linking splicing to innate immunity.
• Dysregulated splicing activation contributes to cancer radioresistance, myelodysplastic syndromes, and viral replication, making it a therapeutic target.
• Neuron-specific splicing is a paradigm of positive regulation, where RNA-binding proteins shift splice site selection to generate neuronal isoforms critical for development.
• CRISPR-based models (knockout, point mutation, knock-in, overexpression) enable causal dissection of splicing regulators and their target exons.
Description
RNA splicing is the process by which introns are removed and exons are joined to form mature mRNA. Positive regulation of RNA splicing (GO:0033120) encompasses any mechanism that activates or increases the frequency, rate or extent of this reaction. This regulation is critical because alternative splicing allows a single gene to produce multiple protein isoforms, expanding proteome diversity and controlling gene expression in development, immunity, and disease. Researchers study positive regulation of splicing to understand how cells respond to stress, how viruses hijack host splicing machinery, and how splicing defects drive cancer and genetic disorders. The term is defined in QuickGO as 'Any process that activates or increases the frequency, rate or extent of RNA splicing,' and it is a biological process that integrates signals from RNA sequence elements, splicing factors, and signaling pathways. Understanding this process is essential for identifying therapeutic targets and for interpreting transcriptomic data in health and disease.
positive regulation of RNA splicing At A Glance
| GO ID | GO:0033120 |
|---|---|
| GO term | positive regulation of RNA splicing |
| Ontology | biological_process |
| Synonym | none |
| Major function | Increases the frequency, rate or extent of RNA splicing, often by recruiting or activating spliceosome components |
| Related processes | RNA splicing, alternative splicing, mRNA processing, spliceosome assembly |
| Key regulators | SR proteins, hnRNPs, PKR, PTBP1, RALY |
| Disease relevance | Cancer, myelodysplastic syndromes, viral infections, neurological disorders |
What Is GO:0033120?
Positive regulation of RNA splicing (GO:0033120) refers to any cellular process that enhances the assembly, activity, or fidelity of the spliceosome, leading to increased splicing of pre-mRNA. This can occur through cis-acting RNA elements that recruit splicing activators, through post-translational modification of splicing factors, or through signaling pathways that alter the availability of splicing components. It is the opposite of negative regulation and is distinct from splicing itself, as it specifically describes the upregulation of splicing frequency or efficiency.
Why Is positive regulation of RNA splicing Important in Cell Biology?
Positive regulation of RNA splicing is fundamental to gene expression because it determines the isoform repertoire of a cell. Dysregulation of this process can lead to aberrant splicing patterns that drive oncogenesis, impair hematopoietic differentiation, or facilitate viral replication. Moreover, the ability to modulate splicing positively is exploited by viruses such as HIV-1 to produce essential viral proteins. Understanding the mechanisms of positive regulation provides opportunities for therapeutic intervention in cancer, genetic diseases, and infectious diseases.
• Controls alternative splicing, enabling proteome diversity from a limited genome.
• Essential for neuronal development and function through neuron-specific splicing programs.
• Plays a key role in immune responses via PKR-mediated splicing activation.
• Contributes to cancer progression and radioresistance, e.g., PTBP1-mediated DNMT3B splicing.
• Involved in myelodysplastic syndromes where splicing factor mutations alter splicing regulation.
• Critical for HIV-1 replication, as viral RNA splicing must be positively regulated for protein production.
• Regulates T cell quiescence and exhaustion through LINE1 splicing.
• Provides targets for antisense oligonucleotides and small molecules that modulate splicing.
• Influences RNA stability and translation via coupling with nonsense-mediated decay.
• Enables rapid cellular responses to stress through stress kinase pathways.
What Happens During positive regulation of RNA splicing?
Cis-acting RNA elements recruit splicing activators
In simple terms: Specific sequences in the RNA act like landing pads for proteins that turn splicing on.
Exonic and intronic splicing enhancers (ESEs and ISEs) are RNA sequences that bind serine/arginine-rich (SR) proteins. This binding stabilizes spliceosome components at nearby splice sites, increasing the rate of exon inclusion. Intragenic RNA elements can also activate the stress kinase PKR, which then positively regulates splicing of cellular and viral mRNAs.
Trans-acting factors and spliceosome assembly
In simple terms: Helper proteins assemble the splicing machine on the RNA and speed up the reaction.
SR proteins and hnRNPs are trans-acting factors that promote or inhibit splice site selection. Positive regulation occurs when activators outcompete repressors or when signaling pathways modify these factors to enhance their activity. For example, PTBP1 interacts with RALY to regulate DNMT3B alternative splicing, enhancing radioresistance in prostate cancer.
Signaling pathways modulate splicing factor activity
In simple terms: Cellular signals can chemically modify splicing proteins to make splicing more efficient.
Stress kinases such as PKR are activated by double-stranded RNA or intragenic elements, leading to phosphorylation of substrates that include splicing factors. This can positively regulate splicing of specific mRNAs, as shown for cellular and viral transcripts. Additionally, neuron-specific splicing is controlled by signaling cascades that alter the localization or activity of RNA-binding proteins.
Coupling with transcription and RNA decay
In simple terms: Splicing is tied to other RNA processing steps, so boosting splicing can affect RNA stability.
Positive regulation of splicing can be coupled to transcription, as the C-terminal domain of RNA polymerase II recruits splicing factors. It can also be linked to nonsense-mediated decay (NMD), where alternative splicing introduces premature stop codons that target transcripts for degradation, as seen for Arp5. LINE1 splicing in non-canonical transcript variants regulates T cell quiescence and exhaustion, illustrating coupling with immune signaling.
Key Genes Involved in GO:0033120 positive regulation of RNA splicing
The following genes and proteins are central to positive regulation of RNA splicing, based on published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| SR proteins (e.g., SRSF1) | Bind exonic splicing enhancers to promote exon inclusion | Key activators of splicing; often overexpressed in cancer |
| hnRNPs (e.g., PTBP1) | Regulate splice site selection; can activate or repress | PTBP1 interacts with RALY to enhance DNMT3B splicing and radioresistance |
| PKR (EIF2AK2) | Stress kinase activated by RNA elements; positively regulates splicing | Links innate immunity to splicing of cellular and viral mRNAs |
| RALY | RNA-binding protein that partners with PTBP1 | Modulates DNMT3B alternative splicing in prostate cancer |
| DNMT3B | DNA methyltransferase with multiple splice variants | Its alternative splicing is positively regulated by PTBP1/RALY |
| Arp5 | Actin-related protein involved in chromatin remodeling | Its expression is regulated by alternative splicing coupled to NMD |
| LINE1 | Retrotransposon that can be spliced in non-canonical variants | Regulates T cell quiescence and exhaustion |
| HIV-1 RNA elements | Viral RNA structures that recruit host splicing factors | Positive regulation of HIV-1 splicing is essential for replication |
| SRSF2 | SR protein involved in spliceosome assembly | Mutations in SRSF2 are common in myelodysplastic syndromes |
| SF3B1 | Core spliceosome component | Mutations in SF3B1 alter splicing and are frequent in MDS |
| U2AF1 | Splicing factor that recognizes 3' splice sites | Mutations in U2AF1 affect splicing regulation in MDS |
| ZRSR2 | Splicing factor involved in 3' splice site recognition | Mutated in MDS, affecting splicing |
| Nova proteins | Neuron-specific splicing regulators | Control neuron-specific alternative splicing |
| PTB (PTBP1/2) | Polypyrimidine tract-binding proteins | Regulate neuronal splicing and cancer-related splicing |
| TIA1 | RNA-binding protein that regulates splicing and translation | Implicated in stress granule and splicing regulation |
| U1 snRNP | Recognizes 5' splice site; essential for spliceosome assembly | Target of positive regulation to enhance splicing |
| U2 snRNP | Binds branch point; key for spliceosome activation | Its recruitment is enhanced by splicing activators |
| SRRM2 | Splicing coactivator | Part of the spliceosome and regulated in cancer |
How Is positive regulation of RNA splicing Regulated?
Positive regulation of RNA splicing is itself regulated at multiple levels. Signaling pathways such as the integrated stress response can activate PKR, which then phosphorylates substrates to enhance splicing of specific mRNAs. Post-translational modifications of SR proteins, including phosphorylation by SRPK and CLK kinases, control their ability to promote splicing. In cancer, overexpression of splicing activators like PTBP1 can shift splicing patterns to favor isoforms that promote radioresistance. Additionally, mutations in splicing factors such as SF3B1, SRSF2, U2AF1, and ZRSR2 in myelodysplastic syndromes alter the normal regulation of splicing, often leading to aberrant positive regulation of specific exons.
positive regulation of RNA splicing and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| PTBP1 | Prostate cancer radioresistance | Knockout or overexpression in prostate cancer cell lines |
| DNMT3B | Cancer, alternative splicing | Point mutation at splice sites to alter isoform ratios |
| SF3B1 | Myelodysplastic syndromes | Knock-in of common SF3B1 mutations in hematopoietic cells |
| SRSF2 | Myelodysplastic syndromes | Knockout or point mutation in leukemia cell lines |
| PKR (EIF2AK2) | Antiviral response, splicing regulation | Knockout in immune cells to study viral splicing |
Cancer and radioresistance
Positive regulation of RNA splicing is hijacked in cancer to produce isoforms that support tumor growth and therapy resistance. For example, PTBP1 interacts with RALY to enhance DNMT3B alternative splicing, promoting radioresistance in prostate cancer. SR proteins are often overexpressed in various cancers, leading to increased inclusion of exons that favor oncogenic pathways.
Myelodysplastic syndromes (MDS)
Mutations in core splicing factors such as SF3B1, SRSF2, U2AF1, and ZRSR2 are common in MDS and lead to altered splicing regulation. These mutations can cause aberrant positive regulation of specific exons, contributing to ineffective hematopoiesis and disease progression.
Viral infections
Viruses like HIV-1 depend on positive regulation of RNA splicing to produce essential viral proteins. HIV-1 RNA elements recruit host splicing factors to enhance splicing of viral transcripts, and this process is a potential target for antiviral therapy. PKR activation by intragenic RNA elements also positively regulates splicing of viral mRNAs as part of the antiviral response.
Neurological disorders
Neuron-specific splicing is critical for neuronal function, and its dysregulation is linked to neurodegenerative diseases. Positive regulation of splicing by RNA-binding proteins such as Nova and PTBP1 controls the inclusion of neuronal exons, and disruptions in these programs can lead to neurological disorders.
From positive regulation of RNA splicing-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of a splicing activator reduce target exon inclusion? | CRISPR knockout of the activator gene in relevant cell line |
| Does a specific point mutation in a splicing factor alter splicing? | CRISPR point mutation knock-in of the mutation |
| Can a splicing enhancer element be sufficient to drive splicing? | Knock-in of the enhancer sequence upstream of a reporter exon |
| What is the effect of overexpressing an SR protein on global splicing? | CRISPR overexpression (e.g., CRISPRa) of the SR protein |
| How does a splicing regulator affect viral replication? | Knockout of the regulator in cells infected with HIV-1 |
| Does alternative splicing coupled to NMD regulate gene expression? | Knockout of NMD factors combined with RNA-seq |
How to Study the positive regulation of RNA splicing Process
| Method | What It Measures | Typical Application |
|---|---|---|
| RNA-seq | Global splicing changes and isoform expression | Identify exons positively regulated by a factor |
| CLIP-seq | Binding sites of RNA-binding proteins | Map where splicing activators bind |
| Minigene assay | Splicing of a reporter exon | Test enhancer elements and factor activity |
| CRISPR screen | Genes that affect splicing when lost or activated | Discover new regulators of positive splicing |
| RT-PCR | Specific splice isoform ratios | Validate splicing changes |
| Ribo-seq | Translation of spliced mRNAs | Link splicing to protein output |
| Proteomics | Protein levels of splicing factors | Assess downstream effects |
| Immunofluorescence | Localization of splicing factors | Study nuclear speckle dynamics |
RNA-seq and splice-aware alignment
RNA sequencing followed by splice-aware alignment (e.g., STAR, HISAT2) and isoform quantification (e.g., rMATS, SUPPA2) is the primary method to measure changes in splicing. It can detect exon inclusion levels and identify targets of positive regulation.
CLIP-seq and RNA immunoprecipitation
Crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) maps binding sites of splicing factors on RNA, revealing how activators are recruited to enhance splicing.
Minigene reporters
Minigene constructs containing specific exons and flanking introns with enhancer elements are used to test whether a sequence or factor positively regulates splicing in vivo.
CRISPR screens
Genome-wide CRISPR knockout or activation screens coupled with splicing reporters can identify novel regulators of positive RNA splicing.
How CRISPR Can Be Used to Study GO:0033120 positive regulation of RNA splicing
Knockout
CRISPR knockout of splicing activators (e.g., PTBP1, SR proteins) can abolish positive regulation of target exons, revealing their necessity. For example, knocking out PTBP1 reduces DNMT3B alternative splicing and reverses radioresistance.
Point Mutation
Point mutations in splice sites or regulatory elements can be introduced to test their role in positive regulation. For instance, mutating a specific residue in SF3B1 that is commonly mutated in MDS can alter splicing patterns.
Knock-in
Knock-in of splicing enhancer elements or tagged splicing factors allows precise tracking of their function. Tagged knock-in of SR proteins enables ChIP or CLIP studies to map their interactions.
Overexpression
CRISPR activation (CRISPRa) or cDNA overexpression can increase levels of splicing activators to study gain-of-function effects on splicing. Overexpressing SRSF1, for example, promotes inclusion of specific exons.
How EDITGENE Supports positive regulation of RNA splicing Research
Researchers studying positive regulation of RNA splicing-related genes often need to determine whether a candidate gene is causally involved in splicing regulation or is merely correlated. EDITGENE provides a comprehensive suite of CRISPR services to enable such causal studies, from knockout to precise point mutations and overexpression.
Contact EDITGENE today to design your custom CRISPR model for positive regulation of RNA splicing research.
Frequently Asked Questions About positive regulation of RNA splicing
What is positive regulation of RNA splicing?
It is any process that increases the frequency, rate or extent of RNA splicing, often by recruiting splicing activators to pre-mRNA.
What genes are involved in positive regulation of RNA splicing?
Key genes include SR proteins (SRSF1), hnRNPs (PTBP1), PKR, RALY, and spliceosome components like SF3B1 and U2AF1.
How does PKR positively regulate splicing?
PKR is activated by intragenic RNA elements and phosphorylates substrates that enhance splicing of cellular and viral mRNAs.
What diseases are linked to dysregulated RNA splicing?
Cancer, myelodysplastic syndromes, viral infections, and neurological disorders are linked to aberrant splicing regulation.
What is the role of PTBP1 in splicing?
PTBP1 interacts with RALY to enhance DNMT3B alternative splicing, promoting radioresistance in prostate cancer.
How can CRISPR be used to study positive regulation of RNA splicing?
CRISPR knockout, point mutation, knock-in, and overexpression allow causal testing of splicing regulators and their target exons.
What methods measure RNA splicing changes?
RNA-seq, CLIP-seq, minigene assays, and RT-PCR are commonly used to quantify splicing changes.
What is the difference between RNA splicing and its positive regulation?
RNA splicing is the reaction itself; positive regulation describes processes that increase its frequency or efficiency.
Which splicing factors are mutated in myelodysplastic syndromes?
SF3B1, SRSF2, U2AF1, and ZRSR2 are frequently mutated in MDS, altering splicing regulation.
How does HIV-1 exploit positive regulation of splicing?
HIV-1 RNA elements recruit host splicing factors to enhance viral RNA splicing, which is essential for replication.
Conclusion
Positive regulation of RNA splicing (GO:0033120) is a critical biological process that controls gene expression by enhancing the efficiency and frequency of splicing. Its dysregulation is implicated in cancer, MDS, viral infections, and neurological disorders, making it a prime target for therapeutic intervention. Advances in CRISPR-based models and high-throughput sequencing are accelerating the discovery of new regulators and their mechanisms. EDITGENE offers end-to-end services to support functional studies of splicing regulation, from knockout to bioinformatics.
References
- 1. Kaempfer R. 2023. Positive Regulation of Splicing of Cellular and Viral mRNA by Intragenic RNA Elements That Activate the Stress Kinase PKR, an Antiviral Mechanism.. Genes (Basel) 14(5) PMID: 37239334
- 2. Morita T et al.. 2023. Regulation of Arp5 expression by alternative splicing coupled to nonsense-mediated RNA decay.. Biochem Biophys Res Commun 657:50-58 PMID: 36977368
- 3. Hakim NH et al.. 2017. Neuron-specific splicing.. Biosci Trends 11(1):16-22 PMID: 28049883
- 4. He H et al.. 2024. PTBP1 Regulates DNMT3B Alternative Splicing by Interacting With RALY to Enhance the Radioresistance of Prostate Cancer.. Adv Sci (Weinh) 11(42):e2405997 PMID: 39287090
- 5. Marasca F et al.. 2022. LINE1 are spliced in non-canonical transcript variants to regulate T cell quiescence and exhaustion.. Nat Genet 54(2):180-193 PMID: 35039641
- 6. Ogawa S. 2019. Genetics of MDS.. Blood 133(10):1049-1059 PMID: 30670442
- 7. Stoltzfus CM. 2009. Chapter 1. Regulation of HIV-1 alternative RNA splicing and its role in virus replication.. Adv Virus Res 74:1-40 PMID: 19698894
- 8. Zheng ZM. 2004. Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression.. J Biomed Sci 11(3):278-94 PMID: 15067211
